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Showing 1 - 200 of 3030 Journals sorted alphabetically
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 20, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 16, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 79, SJR: 1.109, h-index: 94)
Accounting Forum     Hybrid Journal   (Followers: 22, SJR: 0.612, h-index: 27)
Accounting, Organizations and Society     Hybrid Journal   (Followers: 27, SJR: 2.515, h-index: 90)
Achievements in the Life Sciences     Open Access   (Followers: 4)
Acta Anaesthesiologica Taiwanica     Open Access   (Followers: 5, SJR: 0.338, h-index: 19)
Acta Astronautica     Hybrid Journal   (Followers: 303, SJR: 0.726, h-index: 43)
Acta Automatica Sinica     Full-text available via subscription   (Followers: 3)
Acta Biomaterialia     Hybrid Journal   (Followers: 25, SJR: 2.02, h-index: 104)
Acta Colombiana de Cuidado Intensivo     Full-text available via subscription  
Acta de Investigación Psicológica     Open Access   (Followers: 2)
Acta Ecologica Sinica     Open Access   (Followers: 8, SJR: 0.172, h-index: 29)
Acta Haematologica Polonica     Free   (SJR: 0.123, h-index: 8)
Acta Histochemica     Hybrid Journal   (Followers: 3, SJR: 0.604, h-index: 38)
Acta Materialia     Hybrid Journal   (Followers: 196, SJR: 3.683, h-index: 202)
Acta Mathematica Scientia     Full-text available via subscription   (Followers: 5, SJR: 0.615, h-index: 21)
Acta Mechanica Solida Sinica     Full-text available via subscription   (Followers: 9, SJR: 0.442, h-index: 21)
Acta Oecologica     Hybrid Journal   (Followers: 9, SJR: 0.915, h-index: 53)
Acta Otorrinolaringologica (English Edition)     Full-text available via subscription   (Followers: 1)
Acta Otorrinolaringológica Española     Full-text available via subscription   (Followers: 3, SJR: 0.311, h-index: 16)
Acta Pharmaceutica Sinica B     Open Access   (Followers: 2)
Acta Poética     Open Access   (Followers: 4)
Acta Psychologica     Hybrid Journal   (Followers: 21, SJR: 1.365, h-index: 73)
Acta Sociológica     Open Access  
Acta Tropica     Hybrid Journal   (Followers: 5, SJR: 1.059, h-index: 77)
Acta Urológica Portuguesa     Open Access  
Actas Dermo-Sifiliograficas     Full-text available via subscription   (Followers: 4)
Actas Dermo-Sifiliográficas (English Edition)     Full-text available via subscription   (Followers: 3)
Actas Urológicas Españolas     Full-text available via subscription   (Followers: 3, SJR: 0.383, h-index: 19)
Actas Urológicas Españolas (English Edition)     Full-text available via subscription   (Followers: 2)
Actualites Pharmaceutiques     Full-text available via subscription   (Followers: 5, SJR: 0.141, h-index: 3)
Actualites Pharmaceutiques Hospitalieres     Full-text available via subscription   (Followers: 4, SJR: 0.112, h-index: 2)
Acupuncture and Related Therapies     Hybrid Journal   (Followers: 4)
Ad Hoc Networks     Hybrid Journal   (Followers: 11, SJR: 0.967, h-index: 57)
Addictive Behaviors     Hybrid Journal   (Followers: 15, SJR: 1.514, h-index: 92)
Addictive Behaviors Reports     Open Access   (Followers: 5)
Additive Manufacturing     Hybrid Journal   (Followers: 7, SJR: 1.039, h-index: 5)
Additives for Polymers     Full-text available via subscription   (Followers: 20)
Advanced Drug Delivery Reviews     Hybrid Journal   (Followers: 120, SJR: 5.2, h-index: 222)
Advanced Engineering Informatics     Hybrid Journal   (Followers: 11, SJR: 1.265, h-index: 53)
Advanced Powder Technology     Hybrid Journal   (Followers: 16, SJR: 0.739, h-index: 33)
Advances in Accounting     Hybrid Journal   (Followers: 8, SJR: 0.299, h-index: 15)
Advances in Agronomy     Full-text available via subscription   (Followers: 15, SJR: 2.071, h-index: 82)
Advances in Anesthesia     Full-text available via subscription   (Followers: 24, SJR: 0.169, h-index: 4)
Advances in Antiviral Drug Design     Full-text available via subscription   (Followers: 3)
Advances in Applied Mathematics     Full-text available via subscription   (Followers: 6, SJR: 1.054, h-index: 35)
Advances in Applied Mechanics     Full-text available via subscription   (Followers: 10, SJR: 0.801, h-index: 26)
Advances in Applied Microbiology     Full-text available via subscription   (Followers: 21, SJR: 1.286, h-index: 49)
Advances In Atomic, Molecular, and Optical Physics     Full-text available via subscription   (Followers: 16, SJR: 3.31, h-index: 42)
Advances in Biological Regulation     Hybrid Journal   (Followers: 4, SJR: 2.277, h-index: 43)
Advances in Botanical Research     Full-text available via subscription   (Followers: 3, SJR: 0.619, h-index: 48)
Advances in Cancer Research     Full-text available via subscription   (Followers: 26, SJR: 2.215, h-index: 78)
Advances in Carbohydrate Chemistry and Biochemistry     Full-text available via subscription   (Followers: 9, SJR: 0.9, h-index: 30)
Advances in Catalysis     Full-text available via subscription   (Followers: 5, SJR: 2.139, h-index: 42)
Advances in Cellular and Molecular Biology of Membranes and Organelles     Full-text available via subscription   (Followers: 12)
Advances in Chemical Engineering     Full-text available via subscription   (Followers: 24, SJR: 0.183, h-index: 23)
Advances in Child Development and Behavior     Full-text available via subscription   (Followers: 10, SJR: 0.665, h-index: 29)
Advances in Chronic Kidney Disease     Full-text available via subscription   (Followers: 8, SJR: 1.268, h-index: 45)
Advances in Clinical Chemistry     Full-text available via subscription   (Followers: 28, SJR: 0.938, h-index: 33)
Advances in Colloid and Interface Science     Full-text available via subscription   (Followers: 18, SJR: 2.314, h-index: 130)
Advances in Computers     Full-text available via subscription   (Followers: 16, SJR: 0.223, h-index: 22)
Advances in Developmental Biology     Full-text available via subscription   (Followers: 11)
Advances in Digestive Medicine     Open Access   (Followers: 4)
Advances in DNA Sequence-Specific Agents     Full-text available via subscription   (Followers: 5)
Advances in Drug Research     Full-text available via subscription   (Followers: 22)
Advances in Ecological Research     Full-text available via subscription   (Followers: 39, SJR: 3.25, h-index: 43)
Advances in Engineering Software     Hybrid Journal   (Followers: 25, SJR: 0.486, h-index: 10)
Advances in Experimental Biology     Full-text available via subscription   (Followers: 7)
Advances in Experimental Social Psychology     Full-text available via subscription   (Followers: 38, SJR: 5.465, h-index: 64)
Advances in Exploration Geophysics     Full-text available via subscription   (Followers: 3)
Advances in Fluorine Science     Full-text available via subscription   (Followers: 8)
Advances in Food and Nutrition Research     Full-text available via subscription   (Followers: 41, SJR: 0.674, h-index: 38)
Advances in Fuel Cells     Full-text available via subscription   (Followers: 14)
Advances in Genetics     Full-text available via subscription   (Followers: 15, SJR: 2.558, h-index: 54)
Advances in Genome Biology     Full-text available via subscription   (Followers: 11)
Advances in Geophysics     Full-text available via subscription   (Followers: 6, SJR: 2.325, h-index: 20)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 18, SJR: 0.906, h-index: 24)
Advances in Heterocyclic Chemistry     Full-text available via subscription   (Followers: 8, SJR: 0.497, h-index: 31)
Advances in Human Factors/Ergonomics     Full-text available via subscription   (Followers: 22)
Advances in Imaging and Electron Physics     Full-text available via subscription   (Followers: 2, SJR: 0.396, h-index: 27)
Advances in Immunology     Full-text available via subscription   (Followers: 33, SJR: 4.152, h-index: 85)
Advances in Inorganic Chemistry     Full-text available via subscription   (Followers: 9, SJR: 1.132, h-index: 42)
Advances in Insect Physiology     Full-text available via subscription   (Followers: 3, SJR: 1.274, h-index: 27)
Advances in Integrative Medicine     Hybrid Journal   (Followers: 4)
Advances in Intl. Accounting     Full-text available via subscription   (Followers: 4)
Advances in Life Course Research     Hybrid Journal   (Followers: 7, SJR: 0.764, h-index: 15)
Advances in Lipobiology     Full-text available via subscription   (Followers: 1)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 8)
Advances in Marine Biology     Full-text available via subscription   (Followers: 16, SJR: 1.645, h-index: 45)
Advances in Mathematics     Full-text available via subscription   (Followers: 10, SJR: 3.261, h-index: 65)
Advances in Medical Sciences     Hybrid Journal   (Followers: 5, SJR: 0.489, h-index: 25)
Advances in Medicinal Chemistry     Full-text available via subscription   (Followers: 5)
Advances in Microbial Physiology     Full-text available via subscription   (Followers: 4, SJR: 1.44, h-index: 51)
Advances in Molecular and Cell Biology     Full-text available via subscription   (Followers: 21)
Advances in Molecular and Cellular Endocrinology     Full-text available via subscription   (Followers: 10)
Advances in Molecular Toxicology     Full-text available via subscription   (Followers: 6, SJR: 0.324, h-index: 8)
Advances in Nanoporous Materials     Full-text available via subscription   (Followers: 3)
Advances in Oncobiology     Full-text available via subscription   (Followers: 3)
Advances in Organometallic Chemistry     Full-text available via subscription   (Followers: 15, SJR: 2.885, h-index: 45)
Advances in Parallel Computing     Full-text available via subscription   (Followers: 7, SJR: 0.148, h-index: 11)
Advances in Parasitology     Full-text available via subscription   (Followers: 7, SJR: 2.37, h-index: 73)
Advances in Pediatrics     Full-text available via subscription   (Followers: 20, SJR: 0.4, h-index: 28)
Advances in Pharmaceutical Sciences     Full-text available via subscription   (Followers: 14)
Advances in Pharmacology     Full-text available via subscription   (Followers: 13, SJR: 1.718, h-index: 58)
Advances in Physical Organic Chemistry     Full-text available via subscription   (Followers: 7, SJR: 0.384, h-index: 26)
Advances in Phytomedicine     Full-text available via subscription  
Advances in Planar Lipid Bilayers and Liposomes     Full-text available via subscription   (Followers: 3, SJR: 0.248, h-index: 11)
Advances in Plant Biochemistry and Molecular Biology     Full-text available via subscription   (Followers: 8)
Advances in Plant Pathology     Full-text available via subscription   (Followers: 5)
Advances in Porous Media     Full-text available via subscription   (Followers: 4)
Advances in Protein Chemistry     Full-text available via subscription   (Followers: 18)
Advances in Protein Chemistry and Structural Biology     Full-text available via subscription   (Followers: 17, SJR: 1.5, h-index: 62)
Advances in Psychology     Full-text available via subscription   (Followers: 56)
Advances in Quantum Chemistry     Full-text available via subscription   (Followers: 5, SJR: 0.478, h-index: 32)
Advances in Radiation Oncology     Open Access  
Advances in Small Animal Medicine and Surgery     Hybrid Journal   (Followers: 1, SJR: 0.1, h-index: 2)
Advances in Space Research     Full-text available via subscription   (Followers: 332, SJR: 0.606, h-index: 65)
Advances in Structural Biology     Full-text available via subscription   (Followers: 7)
Advances in Surgery     Full-text available via subscription   (Followers: 6, SJR: 0.823, h-index: 27)
Advances in the Study of Behavior     Full-text available via subscription   (Followers: 28, SJR: 1.321, h-index: 56)
Advances in Veterinary Medicine     Full-text available via subscription   (Followers: 14)
Advances in Veterinary Science and Comparative Medicine     Full-text available via subscription   (Followers: 12)
Advances in Virus Research     Full-text available via subscription   (Followers: 5, SJR: 1.878, h-index: 68)
Advances in Water Resources     Hybrid Journal   (Followers: 42, SJR: 2.408, h-index: 94)
Aeolian Research     Hybrid Journal   (Followers: 5, SJR: 0.973, h-index: 22)
Aerospace Science and Technology     Hybrid Journal   (Followers: 304, SJR: 0.816, h-index: 49)
AEU - Intl. J. of Electronics and Communications     Hybrid Journal   (Followers: 8, SJR: 0.318, h-index: 36)
African J. of Emergency Medicine     Open Access   (Followers: 4, SJR: 0.344, h-index: 6)
Ageing Research Reviews     Hybrid Journal   (Followers: 7, SJR: 3.289, h-index: 78)
Aggression and Violent Behavior     Hybrid Journal   (Followers: 390, SJR: 1.385, h-index: 72)
Agri Gene     Hybrid Journal  
Agricultural and Forest Meteorology     Hybrid Journal   (Followers: 15, SJR: 2.18, h-index: 116)
Agricultural Systems     Hybrid Journal   (Followers: 29, SJR: 1.275, h-index: 74)
Agricultural Water Management     Hybrid Journal   (Followers: 36, SJR: 1.546, h-index: 79)
Agriculture and Agricultural Science Procedia     Open Access  
Agriculture and Natural Resources     Open Access   (Followers: 1)
Agriculture, Ecosystems & Environment     Hybrid Journal   (Followers: 48, SJR: 1.879, h-index: 120)
Ain Shams Engineering J.     Open Access   (Followers: 5, SJR: 0.434, h-index: 14)
Air Medical J.     Hybrid Journal   (Followers: 3, SJR: 0.234, h-index: 18)
AKCE Intl. J. of Graphs and Combinatorics     Open Access   (SJR: 0.285, h-index: 3)
Alcohol     Hybrid Journal   (Followers: 9, SJR: 0.922, h-index: 66)
Alcoholism and Drug Addiction     Open Access   (Followers: 5)
Alergologia Polska : Polish J. of Allergology     Full-text available via subscription   (Followers: 1)
Alexandria Engineering J.     Open Access   (Followers: 1, SJR: 0.436, h-index: 12)
Alexandria J. of Medicine     Open Access  
Algal Research     Partially Free   (Followers: 7, SJR: 2.05, h-index: 20)
Alkaloids: Chemical and Biological Perspectives     Full-text available via subscription   (Followers: 3)
Allergologia et Immunopathologia     Full-text available via subscription   (Followers: 1, SJR: 0.46, h-index: 29)
Allergology Intl.     Open Access   (Followers: 5, SJR: 0.776, h-index: 35)
ALTER - European J. of Disability Research / Revue Européenne de Recherche sur le Handicap     Full-text available via subscription   (Followers: 6, SJR: 0.158, h-index: 9)
Alzheimer's & Dementia     Hybrid Journal   (Followers: 45, SJR: 4.289, h-index: 64)
Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring     Open Access   (Followers: 5)
Alzheimer's & Dementia: Translational Research & Clinical Interventions     Open Access   (Followers: 3)
American Heart J.     Hybrid Journal   (Followers: 45, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 47, SJR: 2.063, h-index: 186)
American J. of Emergency Medicine     Hybrid Journal   (Followers: 34, SJR: 0.574, h-index: 65)
American J. of Geriatric Pharmacotherapy     Full-text available via subscription   (Followers: 6, SJR: 1.091, h-index: 45)
American J. of Geriatric Psychiatry     Hybrid Journal   (Followers: 14, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 32, SJR: 8.769, h-index: 256)
American J. of Infection Control     Hybrid Journal   (Followers: 25, SJR: 1.259, h-index: 81)
American J. of Kidney Diseases     Hybrid Journal   (Followers: 31, SJR: 2.313, h-index: 172)
American J. of Medicine     Hybrid Journal   (Followers: 48, SJR: 2.023, h-index: 189)
American J. of Medicine Supplements     Full-text available via subscription   (Followers: 3)
American J. of Obstetrics and Gynecology     Hybrid Journal   (Followers: 174, SJR: 2.255, h-index: 171)
American J. of Ophthalmology     Hybrid Journal   (Followers: 51, SJR: 2.803, h-index: 148)
American J. of Ophthalmology Case Reports     Open Access   (Followers: 2)
American J. of Orthodontics and Dentofacial Orthopedics     Full-text available via subscription   (Followers: 6, SJR: 1.249, h-index: 88)
American J. of Otolaryngology     Hybrid Journal   (Followers: 22, SJR: 0.59, h-index: 45)
American J. of Pathology     Hybrid Journal   (Followers: 23, SJR: 2.653, h-index: 228)
American J. of Preventive Medicine     Hybrid Journal   (Followers: 21, SJR: 2.764, h-index: 154)
American J. of Surgery     Hybrid Journal   (Followers: 32, SJR: 1.286, h-index: 125)
American J. of the Medical Sciences     Hybrid Journal   (Followers: 13, SJR: 0.653, h-index: 70)
Ampersand : An Intl. J. of General and Applied Linguistics     Open Access   (Followers: 5)
Anaerobe     Hybrid Journal   (Followers: 4, SJR: 1.066, h-index: 51)
Anaesthesia & Intensive Care Medicine     Full-text available via subscription   (Followers: 52, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 3)
Anales de Cirugia Vascular     Full-text available via subscription  
Anales de Pediatría     Full-text available via subscription   (Followers: 2, SJR: 0.209, h-index: 27)
Anales de Pediatría (English Edition)     Full-text available via subscription  
Anales de Pediatría Continuada     Full-text available via subscription   (SJR: 0.104, h-index: 3)
Analytic Methods in Accident Research     Hybrid Journal   (Followers: 2, SJR: 2.577, h-index: 7)
Analytica Chimica Acta     Hybrid Journal   (Followers: 38, SJR: 1.548, h-index: 152)
Analytical Biochemistry     Hybrid Journal   (Followers: 154, SJR: 0.725, h-index: 154)
Analytical Chemistry Research     Open Access   (Followers: 7, SJR: 0.18, h-index: 2)
Analytical Spectroscopy Library     Full-text available via subscription   (Followers: 10)
Anesthésie & Réanimation     Full-text available via subscription  
Anesthesiology Clinics     Full-text available via subscription   (Followers: 21, SJR: 0.421, h-index: 40)
Angiología     Full-text available via subscription   (SJR: 0.124, h-index: 9)
Angiologia e Cirurgia Vascular     Open Access  
Animal Behaviour     Hybrid Journal   (Followers: 143, SJR: 1.907, h-index: 126)
Animal Feed Science and Technology     Hybrid Journal   (Followers: 5, SJR: 1.151, h-index: 83)
Animal Reproduction Science     Hybrid Journal   (Followers: 5, SJR: 0.711, h-index: 78)
Annales d'Endocrinologie     Full-text available via subscription   (SJR: 0.394, h-index: 30)
Annales d'Urologie     Full-text available via subscription  
Annales de Cardiologie et d'Angéiologie     Full-text available via subscription   (SJR: 0.177, h-index: 13)
Annales de Chirurgie de la Main et du Membre Supérieur     Full-text available via subscription  
Annales de Chirurgie Plastique Esthétique     Full-text available via subscription   (Followers: 2, SJR: 0.354, h-index: 22)
Annales de Chirurgie Vasculaire     Full-text available via subscription   (Followers: 1)

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Journal Cover Analytical Biochemistry
  [SJR: 0.725]   [H-I: 154]   [154 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3030 journals]
  • Colorimetric detection of microRNA based hybridization chain reaction for
           signal amplification and enzyme for visualization
    • Authors: Na Ying; Taifan Sun; Zhibao Chen; Guangping Song; Bingyao Qi; Shengjun Bu; Xiuwei Sun; Jiayu Wan; Zehong Li
      Pages: 7 - 12
      Abstract: Publication date: 1 July 2017
      Source:Analytical Biochemistry, Volume 528
      Author(s): Na Ying, Taifan Sun, Zhibao Chen, Guangping Song, Bingyao Qi, Shengjun Bu, Xiuwei Sun, Jiayu Wan, Zehong Li
      MicroRNAs (miRNAs) have key roles in gene expression and can be employed as biomarkers for early diagnosis of various diseases, especially cancers. Detection of miRNAs remains challenging and often requires detection platforms. Here, a horseradish peroxidase (HRP)-assisted hybridization chain reaction (HCR) for colorimetric detection of miR-155 was described. In the presence of target miRNA, the capture probe immobilized on the microplate sandwiched the target miR-155 with the 3′ end of the reporter probe. Another exposed part of the RP at the 5'end triggered HCR producing double-stranded DNA polymers with multiple fluorescein isothiocyanates (FITC) for signal amplification. Finally, multiple HRP molecules were immobilized onto the long-range DNA nanostructures through FITC/anti-FITC monoclonal antibody interactions on the microplate for visualization by tetramethylbenzidine/H2O2 system and the colorless substrate turned into the blue product. To obtain accurate data, the absorbance at 450 nm was calculated by microplate reader. The detection limit was 31.8 fM (3.18 amol). Furthermore, this biosensor showed high specificity and was able to discriminate sharply between target miRNA and mismatched sequences. And this approach could be easily applied to the detection of miR-155 in serum sample, thereby ascribing it for a wide application.

      PubDate: 2017-04-25T22:27:25Z
      DOI: 10.1016/j.ab.2017.04.007
      Issue No: Vol. 528 (2017)
  • Enzymatic behavior of laccase following interaction with γ-CD and
           immobilization into PCL nanofibers
    • Authors: M. Fatih Canbolat; Hasan Basri Savas; Fatih Gultekin
      Pages: 13 - 18
      Abstract: Publication date: 1 July 2017
      Source:Analytical Biochemistry, Volume 528
      Author(s): M. Fatih Canbolat, Hasan Basri Savas, Fatih Gultekin
      This study examines the effects of CD use on enzymatic activity, following enzyme immobilization into nanofibers. There is almost no research available on the change in enzyme activity following interaction with cyclodextrin and electrospun nanofiber mats together. Laccase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ-CD addition, and the enzymatic activity of the laccase was analyzed. SEM, XRD, and FTIR analyses were used for the characterization of the resulting structures. Our results showed that cyclodextrin use has a positive effect on the enzyme's activity, and increases its stability. The enzymes treated by cyclodextrin showed activation after complex formation trials, and no activation loss or enzyme denaturation was detected. Our conclusions were supported by the enzyme activity test results, which also showed that immobilization by encapsulation methods gave better activity results than layering methods. Another important finding concerned the laccase's stable characteristics that helped to maintain its enzyme activation after the freeze drying process. Among all test groups, the best activity result was recorded by laccase-γ-CD complex encapsulated PCL nanofibers with 96.48 U/mg.
      Graphical abstract image

      PubDate: 2017-04-25T22:27:25Z
      DOI: 10.1016/j.ab.2017.04.005
      Issue No: Vol. 528 (2017)
  • A sensitive colorimetric determination of cyanuric chloride and its
           activated agarose immobilization resins
    • Authors: Talia Miron; Meir Wilchek
      Pages: 1 - 3
      Abstract: Publication date: 15 June 2017
      Source:Analytical Biochemistry, Volume 527
      Author(s): Talia Miron, Meir Wilchek
      A colorimetric method for determining cyanuric chloride (CC) and for monitoring its polysaccharide gel activation, before and after ligand binding, was developed. The method is based on the reaction of CC or its activated gel with pyridine and barbituric acid or dimethylbarbituric acid. The product formed yields a purple red color with λ max at 595 nm, and an EM value of approximately 300,000 after 1 h at room temperature. Due to its high sensitivity, this method can detect traces of CC (1 μM) under the above conditions. The method is fast, reliable and devoid of any side reactions.

      PubDate: 2017-04-12T12:44:03Z
      DOI: 10.1016/j.ab.2017.03.026
      Issue No: Vol. 527 (2017)
  • A novel voltammetric sensor for nevirapine, based on modified graphite
           electrode by MWCNs/poly(methylene blue)/gold nanoparticle
    • Authors: Mohammad Bagher Gholivand; Elahe Ahmadi; Mozhdeh Haseli
      Pages: 4 - 12
      Abstract: Publication date: 15 June 2017
      Source:Analytical Biochemistry, Volume 527
      Author(s): Mohammad Bagher Gholivand, Elahe Ahmadi, Mozhdeh Haseli
      In the present study, a graphite electrode (GE) modified by conductive film (containing functionalized multi-walled carbon nanotubes (f-MWCNTs), poly methylene blue p(MB) and gold nanoparticles (AuNPs)) was introduced for determination of nevirapine (NVP) as an anti-HIV drug by applying the differential pulse anodic stripping voltammetry (DPASV) technique. Modification of the electrode was investigated by scanning electron microscopy (SEM) and impedance electrochemical spectroscopy (EIS). All electrochemical effective parameters on detection of NVP were optimized and the oxidation peak current of drug was used for its monitoring. The obtained results confirmed that the oxidation peak currents increased linearly by increasing in NVP concentrations in the range of 0.1–50 μM and a detection limit of 53 nM was achieved. The proposed sensor (AuNPs/p(MB)/f-MWCNTs/GE) was successfully applied for the determination of NVP in blood serum and pharmaceutical samples. It revealed the excellent stability, repeatability and reproducibility as well.

      PubDate: 2017-04-12T12:44:03Z
      DOI: 10.1016/j.ab.2017.03.018
      Issue No: Vol. 527 (2017)
  • The use of flow cytometry to examine calcium signalling by TRPV1 in mixed
           cell populations
    • Authors: Bakri M. Assas; Wesam H. Abdulaal; Majed H. Wakid; Haytham A. Zakai; J. Miyan; J.L. Pennock
      Pages: 13 - 19
      Abstract: Publication date: 15 June 2017
      Source:Analytical Biochemistry, Volume 527
      Author(s): Bakri M. Assas, Wesam H. Abdulaal, Majed H. Wakid, Haytham A. Zakai, J. Miyan, J.L. Pennock
      Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment.

      PubDate: 2017-04-12T12:44:03Z
      DOI: 10.1016/j.ab.2017.03.025
      Issue No: Vol. 527 (2017)
  • A yeast growth assay to characterize plant poly(ADP-ribose) polymerase
           (PARP) proteins and inhibitors
    • Authors: Dagmar Rissel; Peter Paul Heym; Edgar Peiter
      Pages: 20 - 23
      Abstract: Publication date: 15 June 2017
      Source:Analytical Biochemistry, Volume 527
      Author(s): Dagmar Rissel, Peter Paul Heym, Edgar Peiter
      Poly(ADP-ribose) polymerases (PARPs) have been implicated in responses of plants to DNA damage and numerous stresses, whereby the mechanistic basis of the interference is often unclear. Therefore, the identification of specific inhibitors and potential interactors of plant PARPs is desirable. For this purpose, we established an assay based on heterologous expression of PARP genes from the model plant Arabidopsis thaliana in yeast. Expression of AtPARPs caused an inhibition of yeast growth to different extent, which was alleviated by inhibitors targeted at human PARPs. This assay provides a fast and simple means to identify target proteins and pharmacological inhibitors of AtPARP1.

      PubDate: 2017-04-19T18:31:57Z
      DOI: 10.1016/j.ab.2017.04.002
      Issue No: Vol. 527 (2017)
  • SucStruct: Prediction of succinylated lysine residues by using structural
           properties of amino acids
    • Authors: Yosvany López; Abdollah Dehzangi; Sunil Pranit Lal; Ghazaleh Taherzadeh; Jacob Michaelson; Abdul Sattar; Tatsuhiko Tsunoda; Alok Sharma
      Pages: 24 - 32
      Abstract: Publication date: 15 June 2017
      Source:Analytical Biochemistry, Volume 527
      Author(s): Yosvany López, Abdollah Dehzangi, Sunil Pranit Lal, Ghazaleh Taherzadeh, Jacob Michaelson, Abdul Sattar, Tatsuhiko Tsunoda, Alok Sharma
      Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. In contrast to the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming. Therefore, the development of computational tools for accurately predicting succinylated lysines is an urgent necessity. To date, several approaches have been proposed but their sensitivity has been reportedly poor. In this paper, we propose an approach that utilizes structural features of amino acids to improve lysine succinylation prediction. Succinylated and non-succinylated lysines were first retrieved from 670 proteins and characteristics such as accessible surface area, backbone torsion angles and local structure conformations were incorporated. We used the k-nearest neighbors cleaning treatment for dealing with class imbalance and designed a pruned decision tree for classification. Our predictor, referred to as SucStruct (Succinylation using Structural features), proved to significantly improve performance when compared to previous predictors, with sensitivity, accuracy and Mathew's correlation coefficient equal to 0.7334–0.7946, 0.7444–0.7608 and 0.4884–0.5240, respectively.

      PubDate: 2017-04-19T18:31:57Z
      DOI: 10.1016/j.ab.2017.03.021
      Issue No: Vol. 527 (2017)
  • Determination of thiol-to-protein ratio and drug-to-antibody ratio by
           in-line size exclusion chromatography with post-column reaction
    • Authors: Kenichiro Furuki; Toshimasa Toyo'oka
      Pages: 33 - 44
      Abstract: Publication date: Available online 18 April 2017
      Source:Analytical Biochemistry
      Author(s): Kenichiro Furuki, Toshimasa Toyo'oka
      An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 μM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.

      PubDate: 2017-04-19T18:31:57Z
      DOI: 10.1016/j.ab.2017.04.008
      Issue No: Vol. 527 (2017)
  • Characterization of succinimide stability during trypsin digestion for
           LC-MS analysis
    • Authors: Christine Nowak; Gomathinayagam Ponniah; Alyssa Neill; Hongcheng Liu
      Pages: 1 - 8
      Abstract: Publication date: 1 June 2017
      Source:Analytical Biochemistry, Volume 526
      Author(s): Christine Nowak, Gomathinayagam Ponniah, Alyssa Neill, Hongcheng Liu
      LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5.0 which was previously determined to be optimal for the detection of succinimide. The same procedure was able to simultaneously generate approximately 3% succinimide. Using the optimized procedure, it was also found that two aspartate residues that are followed by glycine residues in the conserved Fc region of a recombinant monoclonal antibody were not prone to isomerization. On the other hand, an aspartate residue followed by a glycine in the heavy chain variable domain was highly susceptible to isomerization. Interestingly, the antibody containing the succinimide eluted from an SEC column after the monomer peak.

      PubDate: 2017-03-22T08:29:39Z
      DOI: 10.1016/j.ab.2017.03.005
      Issue No: Vol. 526 (2017)
  • Analysis of short-chain fatty acids in human feces: A scoping review
    • Authors: Maša Primec; Dušanka Mičetić-Turk; Tomaž Langerholc
      Pages: 9 - 21
      Abstract: Publication date: Available online 12 March 2017
      Source:Analytical Biochemistry
      Author(s): Maša Primec, Dušanka Mičetić-Turk, Tomaž Langerholc
      Short-chain fatty acids (SCFAs) play a crucial role in maintaining homeostasis in humans, therefore the importance of a good and reliable SCFAs analytical detection has raised a lot in the past few years. The aim of this scoping review is to show the trends in the development of different methods of SCFAs analysis in feces, based on the literature published in the last eleven years in all major indexing databases. The search criteria included analytical quantification techniques of SCFAs in different human clinical and in vivo studies. SCFAs analysis is still predominantly performed using gas chromatography (GC), followed by high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and capillary electrophoresis (CE). Performances, drawbacks and advantages of these methods are discussed, especially in the light of choosing a proper pretreatment, as feces is a complex biological material. Further optimization to develop a simple, cost effective and robust method for routine use is needed.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.03.007
      Issue No: Vol. 526 (2017)
  • Novel method for measurement of heparin anticoagulant activity using SPR
    • Authors: Jing Zhao; Xinyue Liu; Anju Malhotra; Quanhong Li; Fuming Zhang; Robert J. Linhardt
      Pages: 39 - 42
      Abstract: Publication date: 1 June 2017
      Source:Analytical Biochemistry, Volume 526
      Author(s): Jing Zhao, Xinyue Liu, Anju Malhotra, Quanhong Li, Fuming Zhang, Robert J. Linhardt
      A novel method has been developed for the easy measurement of heparin's anticoagulant activity using surface plasmon resonance. The anticoagulant activity of target heparin was evaluated by measuring the competitive antithrombin III binding of analyte heparin in the solution phase and USP heparin immobilized on chip surface. Heparins, obtained from different animal sources, and low molecular weight heparins were analyzed. The results were reproducible and correlated well with the results of chromogenic assays (correlation coefficient r = 0.98 for anti-Xa and r = 0.94 for anti-IIa). This protocol provides many advantages, significantly minimizing time, cost and the complications of chromogenic assay methods.

      PubDate: 2017-03-22T08:29:39Z
      DOI: 10.1016/j.ab.2017.03.013
      Issue No: Vol. 526 (2017)
  • Simple quantitative PCR analysis for allelic Pten loss in tumor
    • Authors: Xingping Wu; Ailin Zhu; Xiaowu Pang; Guiqin Xie; Xinbin Gu
      Pages: 50 - 57
      Abstract: Publication date: Available online 18 March 2017
      Source:Analytical Biochemistry
      Author(s): Xingping Wu, Ailin Zhu, Xiaowu Pang, Guiqin Xie, Xinbin Gu
      We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR. As a method to detect both mutation and allele-specific gene loss, our approach is less subject to the variability of sample amount that are often very limited in clinical analysis. Since conventional PCR is easy to be carried out, our method simplifies the workflow in any laboratory and would provide significant advantages for simplicity to quantify allele-specific gene loss.

      PubDate: 2017-03-22T08:29:39Z
      DOI: 10.1016/j.ab.2017.03.016
      Issue No: Vol. 526 (2017)
  • Pulsed-field gel electrophoresis does not break E. coli chromosome
           undergoing excision repair after UV irradiation
    • Authors: Sharik R. Khan; Andrei Kuzminov
      Pages: 66 - 68
      Abstract: Publication date: 1 June 2017
      Source:Analytical Biochemistry, Volume 526
      Author(s): Sharik R. Khan, Andrei Kuzminov
      We showed before that long linear DNA molecules containing single-strand interruptions and undergoing pulsed-field gel electrophoresis (PFGE) tend to break into subfragments (electrophoretic nick instability). Here we show that circular chromosomal DNA with single-strand interruptions remains in the wells during PFGE. This means that the presence of nicks in immobile circular DNA is not enough to break this DNA during PFGE. In other words, under the conditions of our study, the artifactual conversion of nicks into double-strand breaks that we detect in linear DNA does not contribute to the overall level of chromosomal fragmentation, as measured by PFGE.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2017.03.019
      Issue No: Vol. 526 (2017)
  • Tips on the analysis of phosphatidic acid by the fluorometric coupled
           enzyme assay
    • Authors: Azam Hassaninasab; Gil-Soo Han; George M. Carman
      Pages: 69 - 70
      Abstract: Publication date: 1 June 2017
      Source:Analytical Biochemistry, Volume 526
      Author(s): Azam Hassaninasab, Gil-Soo Han, George M. Carman
      The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2017.03.020
      Issue No: Vol. 526 (2017)
  • Facile preparation of a fluorescent probe to detect the cellular ability
           of nucleotide excision repair
    • Authors: Hana Tawarahara; Isao Kuraoka; Shigenori Iwai
      Pages: 71 - 74
      Abstract: Publication date: 1 June 2017
      Source:Analytical Biochemistry, Volume 526
      Author(s): Hana Tawarahara, Isao Kuraoka, Shigenori Iwai
      We previously developed a method to detect the cellular ability of nucleotide excision repair, which functions to remove UV-induced lesions in DNA, using a plasmid-type fluorescent probe. A drawback to the popular use of this method was that the oligonucleotide containing the (6–4) photoproduct, which was used as a primer in the plasmid preparation, must be synthesized chemically. In this study, we prepared the probe using a post-synthetically UV-irradiated oligonucleotide as the primer. Transfection of cells demonstrated that this probe detected the repair ability of the cells in the same manner as the original probe.

      PubDate: 2017-04-05T09:45:27Z
      DOI: 10.1016/j.ab.2017.03.023
      Issue No: Vol. 526 (2017)
  • Quantitation of plasmatic lysosphingomyelin and lysosphingomyelin-509 for
           differential screening of Niemann-Pick A/B and C diseases
    • Authors: L. Kuchar; J. Sikora; M.E. Gulinello; H. Poupetova; A. Lugowska; V. Malinova; H. Jahnova; B. Asfaw; J. Ledvinova
      Pages: 73 - 77
      Abstract: Publication date: 15 May 2017
      Source:Analytical Biochemistry, Volume 525
      Author(s): L. Kuchar, J. Sikora, M.E. Gulinello, H. Poupetova, A. Lugowska, V. Malinova, H. Jahnova, B. Asfaw, J. Ledvinova
      Acid sphingomyelinase deficiency (ASMd, Niemann-Pick disease A/B) and Niemann-Pick type C disease (NPC) share core clinical symptoms. Initial diagnostic discrimination of these two rare lysosomal storage diseases is thus difficult. As sphingomyelin accumulates in ASMd as well as NPC, lysosphingomyelin (sphingosylphosphorylcholine) and its m/z 509 analog were suggested as biomarkers for both diseases. Herein we present results of simultaneous LC-ESI-MS/MS measurements of lysosphingomyelin and lysosphingomyelin 509 in plasma and dried blood spots (DBS) collected from ASMd and NPC patients and suggest that the plasma but not DBS levels of the two analytes allow differential biochemical screening of ASMd and NPC.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.02.019
      Issue No: Vol. 525 (2017)
  • High affinity truncated DNA aptamers for the development of fluorescence
           based progesterone biosensors
    • Authors: Hani A. Alhadrami; Raja Chinnappan; Shimaa Eissa; Anas Abdel Rahamn; Mohammed Zourob
      Pages: 78 - 84
      Abstract: Publication date: 15 May 2017
      Source:Analytical Biochemistry, Volume 525
      Author(s): Hani A. Alhadrami, Raja Chinnappan, Shimaa Eissa, Anas Abdel Rahamn, Mohammed Zourob
      Aptamers have shown a number of potential applications in sensing and therapeutic due to the high affinity and specificity towards their target molecules. Not all the nucleotides in the full length aptamers are involved in the binding with their targets. The non-binding domain of the aptamer may affect the binding affinity of the aptamer-target complex. Mapping the aptamer binding region could increase the affinity and the specificity. In this paper, we designed aptamer-based fluorescence sensors from a truncated progesterone (P4) aptamer. Then, fluorescein and quencher labelled aptamer complementary oligonucleotide sequences were hybridized to the truncated aptamer at different sites to form duplex structures. We used fluorescence-quencher pair displacement assay upon progesterone binding for the determination of P4. One of the truncated sequences has shown high binding affinity with 16 fold increase in the dissociation constant, Kd (2.1 nM) compared to the original aptamer. The aptasensor was highly selective for P4 against similar compounds such as 17-β estradiol, bisphenol-A and vitamin D. The sensor has been applied for the detection of P4 in spiked tap water and in urine samples showing good recovery. This new developed aptamer-based fluorescence biosensor can be applied in food, pharmaceutical, and clinical industries.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.02.014
      Issue No: Vol. 525 (2017)
  • Development of competitive ELISA for the detection of bovine serum albumin
           using single-chain variable fragments
    • Authors: Yuan Liu; Manman Lin; Xifeng Zhang; Xiaodan Hu; Jieru Lin; Jia Hao; Dan He; Xiao Zhang; Chongxin Xu; Jianfeng Zhong; Yajing Xie; Cunzheng Zhang; Xianjin Liu
      Pages: 89 - 91
      Abstract: Publication date: 15 May 2017
      Source:Analytical Biochemistry, Volume 525
      Author(s): Yuan Liu, Manman Lin, Xifeng Zhang, Xiaodan Hu, Jieru Lin, Jia Hao, Dan He, Xiao Zhang, Chongxin Xu, Jianfeng Zhong, Yajing Xie, Cunzheng Zhang, Xianjin Liu
      Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10−8 M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 μg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

      PubDate: 2017-04-12T12:44:03Z
      DOI: 10.1016/j.ab.2017.03.003
      Issue No: Vol. 525 (2017)
  • A colorimetric aptasensor for sulfadimethoxine detection based on
           peroxidase-like activity of graphene/nickel@palladium hybrids
    • Authors: Aicheng Wang; Huimin Zhao; Xiaochi Chen; Bing Tan; Yaobin Zhang; Xie Quan
      Pages: 92 - 99
      Abstract: Publication date: 15 May 2017
      Source:Analytical Biochemistry, Volume 525
      Author(s): Aicheng Wang, Huimin Zhao, Xiaochi Chen, Bing Tan, Yaobin Zhang, Xie Quan
      A sensitive, rapid and label-free colorimetric aptasensor for sulfadimethoxine (SDM) detection was developed based on the tunable peroxidase-like activity of graphene/nickel@palladium nanoparticle (Gr/Ni@Pd) hybrids. The addition of the SDM aptamer could inhibit the peroxidase-like catalytic activity of the hybrids. However, the target SDM and aptamer could be triggered tightly and recover the catalytic activity of the Gr/Ni@Pd hybrids. Due to the peroxidase-like catalytic activity, Gr/Ni@Pd could catalyze the decomposition of H2O2 with releasing hydroxyl radicals which further oxidized reagent 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) to oxTMB accompanied with a colorless-to-blue color change. The original color change could be applied to obtain quantitative detection of SDM, due to the relationship between the concentration of the target and the color difference. As a result, this approach performed a linear response for SDM from 1 to 500 ng/mL with a limit detection of 0.7 ng/mL (S/N = 3) under the optimized conditions and realized the detection of SDM in spiked lake water samples. Therefore, this colorimetric aptasensor was an alternative assay for SDM detection in real water. Moreover, with its design principle, this work might be applied to detecting other small molecule by employing appropriate aptamer.

      PubDate: 2017-04-12T12:44:03Z
      DOI: 10.1016/j.ab.2017.03.006
      Issue No: Vol. 525 (2017)
  • predCar-site: Carbonylation sites prediction in proteins using support
           vector machine with resolving data imbalanced issue
    • Authors: Md. Al Mehedi Hasan; Jinyan Li; Shamim Ahmad; Md. Khademul Islam Molla
      Pages: 107 - 113
      Abstract: Publication date: 15 May 2017
      Source:Analytical Biochemistry, Volume 525
      Author(s): Md. Al Mehedi Hasan, Jinyan Li, Shamim Ahmad, Md. Khademul Islam Molla
      The carbonylation is found as an irreversible post-translational modification and considered a biomarker of oxidative stress. It plays major role not only in orchestrating various biological processes but also associated with some diseases such as Alzheimer's disease, diabetes, and Parkinson's disease. However, since the experimental technologies are costly and time-consuming to detect the carbonylation sites in proteins, an accurate computational method for predicting carbonylation sites is an urgent issue which can be useful for drug development. In this study, a novel computational tool termed predCar-Site has been developed to predict protein carbonylation sites by (1) incorporating the sequence-coupled information into the general pseudo amino acid composition, (2) balancing the effect of skewed training dataset by Different Error Costs method, and (3) constructing a predictor using support vector machine as classifier. This predCar-Site predictor achieves an average AUC (area under curve) score of 0.9959, 0.9999, 1, and 0.9997 in predicting the carbonylation sites of K, P, R, and T, respectively. All of the experimental results along with AUC are found from the average of 5 complete runs of the 10-fold cross-validation and those results indicate significantly better performance than existing predictors. A user-friendly web server of predCar-Site is available at

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2017.03.008
      Issue No: Vol. 525 (2017)
  • Editorial for Special Issue on lipid methodology
    • Authors: Howard Goldfine; Ziqiang Guan
      Pages: 1 - 2
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Howard Goldfine, Ziqiang Guan

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2017.02.008
      Issue No: Vol. 524 (2017)
  • Regioisomeric and enantiomeric analysis of triacylglycerols
    • Authors: Tomáš Řezanka; Karolína Pádrová; Karel Sigler
      Pages: 3 - 12
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Tomáš Řezanka, Karolína Pádrová, Karel Sigler
      A survey of useful methods for separation and identification of regioisomers and enantiomers of triacylglycerols. Gas chromatography, gas chromatography-mass spectrometry, 13C NMR determination of regioisomers by enzymatic methods, and supercritical fluid chromatography are briefly surveyed, whereas a detailed description is given of the analysis of triacylglycerols by liquid chromatography, especially with silver ion (Ag+; argentation), and nonaqueous reversed phase liquid chromatography. Special attention is paid to chiral chromatography. Details of mass spectrometry of triacylglycerols are also described, especially the identification of important triacylglycerol ions such as [M + H-RCOOH]+ in atmospheric pressure chemical ionization mass spectra.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2016.05.028
      Issue No: Vol. 524 (2017)
  • Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and
           relatives in biological samples: Analytical and biological challenges
    • Authors: Dimitrios Tsikas
      Pages: 13 - 30
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Dimitrios Tsikas
      Malondialdehyde (MDA), 4-hydroxy-nonenal (HNE) and the F2-isoprostane 15(S)-8-iso-prostaglandin F2α (15(S)-8-iso-PGF2α) are the best investigated products of lipid peroxidation. MDA, HNE and 15(S)-8-iso-PGF2α are produced from polyunsaturated fatty acids (PUFAs) both by chemical reactions and by reactions catalyzed by enzymes. 15(S)-8-iso-PGF2α and other F2-isoprostanes are derived exclusively from arachidonic acid (AA). The number of PUFAs that may contribute to MDA and HNE is much higher. MDA is the prototype of the so called thiobarbituric acid reactive substances (TBARS). MDA, HNE and 15(S)-8-iso-PGF2α are the most frequently measured biomarkers of oxidative stress, namely of lipid peroxidation. In many diseases, higher concentrations of MDA, HNE and 15(S)-8-iso-PGF2α are measured in biological samples as compared to health. Therefore, elevated oxidative stress is generally regarded as a pathological condition. Decreasing the concentration of biomarkers of oxidative stress by changing life style, by nutritional intake of antioxidants or by means of drugs is generally believed to be beneficial to health. Reliable assessment of oxidative stress by measuring MDA, HNE and 15(S)-8-iso-PGF2α in biological fluids is highly challenging for two important reasons: Because of the duality of oxidative stress, i.e., its origin from chemical and enzymatic reactions, and because of pre-analytical and analytical issues. This article focuses on these key issues. It reviews reported analytical methods and their principles for the quantitative measurement of MDA, HNE and 15(S)-8-iso-PGF2α in biological samples including plasma and urine, and critically discusses their biological and biomedical outcome which is rarely crystal clear and free of artefacts.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2016.10.021
      Issue No: Vol. 524 (2017)
  • Simultaneous GC-MS/MS measurement of malondialdehyde and
           4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine
    • Authors: Dimitrios Tsikas; Sabine Rothmann; Jessica Y. Schneider; Frank-Mathias Gutzki; Bibiana Beckmann; Jürgen C. Frölich
      Pages: 31 - 44
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Dimitrios Tsikas, Sabine Rothmann, Jessica Y. Schneider, Frank-Mathias Gutzki, Bibiana Beckmann, Jürgen C. Frölich
      Here, we report the simultaneous derivatization and quantification of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) in human plasma by GC-MS/MS using [1,3-2H2]-MDA (d2-MDA) and [9,9,9-2H3]-HNE (d3-HNE) as the internal standards, respectively. MDA, d2-MDA, HNE and d3-HNE were converted to their pentafluorobenzyl oximes (PFBOX) by pentafluorobenzyl hydroxylamine. Subsequently, the hydroxyl groups of the PFBOX of HNE and d3-HNE were trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane. GC-MS/MS analyses were performed in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring the mass transitions m/z 442 to m/z 243 for MDA, m/z 444 to m/z 244 for d2-MDA, m/z 403 → m/z 283 for HNE and m/z 406 → m/z 286 for d3-HNE. The method was applied to measure MDA and HNE in plasma of patients suffering from coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD) before and after oral supplementation of L-arginine (3 g/day) or placebo for 3 (CAD and PAOD) and 6 months (PAOD). All plasma samples were analyzed after completion of the studies. Our results revealed that storage of plasma samples (at −80 °C) leads to lower MDA and HNE plasma concentrations in the plasma samples that were collected at the end of the studies as compared to those collected at the begin of the studies. Based on MDA and HNE measurements in plasma, L-arginine did not influence lipid peroxidation in CAD and PAOD patients. Long-term studies on lipid peroxidation are best performed by measuring oxidative stress biomarkers such as MDA and/or HNE in plasma samples immediately after their collection. Long-term storage of plasma samples even at −80 °C is not recommended.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2016.08.009
      Issue No: Vol. 524 (2017)
  • Advances and unresolved challenges in the structural characterization of
           isomeric lipids
    • Authors: Sarah E. Hancock; Berwyck L.J. Poad; Amani Batarseh; Sarah K. Abbott; Todd W. Mitchell
      Pages: 45 - 55
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Sarah E. Hancock, Berwyck L.J. Poad, Amani Batarseh, Sarah K. Abbott, Todd W. Mitchell
      As the field of lipidomics grows and its application becomes wide and varied it is important that we don't forget its foundation, i.e. the identification and measurement of molecular lipids. Advances in liquid chromatography and the emergence of ion mobility as a useful tool in lipid analysis are allowing greater separation of lipid isomers than ever before. At the same time, novel ion activation techniques, such as ozone-induced dissociation, are pushing lipid structural characterization by mass spectrometry to new levels. Nevertheless, the quantitative capacity of these techniques is yet to be proven and further refinements are required to unravel the high level of lipid complexity found in biological samples. At present there is no one technique capable of providing full structural characterization of lipids from a biological sample. There are however, numerous techniques now available (as discussed in this review) that could be deployed in a targeted approach. Moving forward, the combination of advanced separation and ion activation techniques is likely to provide mass spectrometry-based lipidomics with its best opportunity to achieve complete molecular-level lipid characterization and measurement from complex mixtures.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2016.09.014
      Issue No: Vol. 524 (2017)
  • Cholesterolomics: An update
    • Authors: William J. Griffiths; Jonas Abdel-Khalik; Eylan Yutuc; Alwena H. Morgan; Ian Gilmore; Thomas Hearn; Yuqin Wang
      Pages: 56 - 67
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): William J. Griffiths, Jonas Abdel-Khalik, Eylan Yutuc, Alwena H. Morgan, Ian Gilmore, Thomas Hearn, Yuqin Wang
      Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D3, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this short article we will concentrate our attention on intermediates in bile acid biosynthesis pathways, in particular oxysterols and cholestenoic acids. These molecular classes are implicated in the aetiology of a diverse array of diseases including autoimmune disease, Parkinson's disease, motor neuron disease, breast cancer, the lysosomal storage disease Niemann-Pick type C and the autosomal recessive disorder Smith-Lemli-Opitz syndrome. Mass spectrometry (MS) is the dominant technology for sterol analysis including both gas-chromatography (GC)-MS and liquid chromatography (LC)-MS and more recently matrix-assisted laser desorption/ionisation (MALDI)-MS for tissue imaging studies. Here we will discuss exciting biological findings and recent analytical improvements.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2017.01.009
      Issue No: Vol. 524 (2017)
  • Metabolic tracing of monoacylglycerol acyltransferase-2 activity
           in vitro and in vivo
    • Authors: Jenson Qi; Wensheng Lang; Margery A. Connelly; Fuyong Du; Yin Liang; Gary W. Caldwell; Tonya Martin; Michael K. Hansen; Gee-Hong Kuo; Michael D. Gaul; Alessandro Pocai; Seunghun Lee
      Pages: 68 - 75
      Abstract: Publication date: 1 May 2017
      Source:Analytical Biochemistry, Volume 524
      Author(s): Jenson Qi, Wensheng Lang, Margery A. Connelly, Fuyong Du, Yin Liang, Gary W. Caldwell, Tonya Martin, Michael K. Hansen, Gee-Hong Kuo, Michael D. Gaul, Alessandro Pocai, Seunghun Lee
      Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus® and 0.59 g/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and U13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.

      PubDate: 2017-03-29T09:34:49Z
      DOI: 10.1016/j.ab.2016.09.017
      Issue No: Vol. 524 (2017)
  • Capillary electrochromatography immunoassay for alpha-fetoprotein based on
           poly(guanidinium ionic liquid) monolithic material
    • Authors: Cuicui Liu; Qiliang Deng; Guozhen Fang; Meng Dang; Shuo Wang
      Abstract: Publication date: Available online 25 April 2017
      Source:Analytical Biochemistry
      Author(s): Cuicui Liu, Qiliang Deng, Guozhen Fang, Meng Dang, Shuo Wang
      Alpha-fetoprotein (AFP) is widely used as a tumor marker for the serum diagnosis of primary hepatoma. Sensitive detection of AFP level plays an important role in the early diagnosis of disease and highly reliable prediction. In this study, a novel non-competitive immunoassay (IA) based on poly(guanidinium ionic liquid) monolithic material was developed for detecting ultra trace levels of AFP in capillary electrochromatography (CEC) mode. The AFP was mixed with an excess amount of fluorescently labeled antibody. After incubation, the immunocomplex was separated from the free labeled antibody and detected by CEC coupled with laser-induced fluorescence detector. Under the optimized conditions, the developed CEC-IA performed a low detection limit of 0.05 μg L−1 (S/N = 3) and a wide linearity ranging from 0.1 to 1000 μg L−1 for AFP, which can be largely attributed to the high separation and enrichment efficiency of poly(guanidinium ionic liquid) monolithic material for the targets. The application of this method was demonstrated by determining AFP in human serum.

      PubDate: 2017-04-25T22:27:25Z
      DOI: 10.1016/j.ab.2017.04.014
  • Characterization of binding and quantification of human autoantibodies to
           PDGFRα using a biosensor-based approach
    • Authors: Gianluca Moroncini; Massimiliano Cuccioloni; Matteo Mozzicafreddo; Katarzyna Natalia Pozniak; Antonella Grieco; Chiara Paolini; Cecilia Tonnini; Tatiana Spadoni; Silvia Svegliati; Ada Funaro; Mauro Angeletti; Armando Gabrielli
      Abstract: Publication date: Available online 24 April 2017
      Source:Analytical Biochemistry
      Author(s): Gianluca Moroncini, Massimiliano Cuccioloni, Matteo Mozzicafreddo, Katarzyna Natalia Pozniak, Antonella Grieco, Chiara Paolini, Cecilia Tonnini, Tatiana Spadoni, Silvia Svegliati, Ada Funaro, Mauro Angeletti, Armando Gabrielli
      Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20–4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.
      Graphical abstract image

      PubDate: 2017-04-25T22:27:25Z
      DOI: 10.1016/j.ab.2017.04.011
  • Novel method to rapidly and efficiently lyse Escherichia coli for the
           isolation of recombinant protein
    • Authors: Himanshu Joshi; Vikas Jain
      Abstract: Publication date: Available online 18 April 2017
      Source:Analytical Biochemistry
      Author(s): Himanshu Joshi, Vikas Jain
      Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity. We report here a novel method to lyse E. coli, which is rapid, and results in high yield of isolated protein. Here, we have carried out intracellular expression of lysozyme domain (LD) of mycobacteriophage D29 endolysin. LD remains non-toxic until chloroform is added into the culture medium that permeabilizes bacterial cell membrane and allows the diffusion of LD to the peptidoglycan layer causing latter's degradation ensuing cell lysis. Our method efficiently lyses E. coli in short duration. As a proof-of-concept, we demonstrate large scale isolation and purification of α subunit of E. coli RNA polymerase and GFP, when they are co-expressed with LD. We believe that our method will be adopted easily in high-throughput as well as large scale protein isolation experiments.

      PubDate: 2017-04-19T18:31:57Z
      DOI: 10.1016/j.ab.2017.04.009
  • Sensitive detection of L-5-hydroxytryptophan based on molecularly
           imprinted polymers with graphene amplification
    • Authors: Lu Chen; Hui-Ting Lian; Xiang-Ying Sun; Bin Liu
      Abstract: Publication date: Available online 19 March 2017
      Source:Analytical Biochemistry
      Author(s): Lu Chen, Hui-Ting Lian, Xiang-Ying Sun, Bin Liu
      A novel electrochemical sensor was presented for the determination of L-5-hydroxytryptophan (L-5-HTP) based on a graphene-chitosan molecularly imprinted film modified on the surface of glassy carbon electrode (GR-MIP/GCE). The morphology and composition of the imprinted film were observed in field emission scanning electron microscopy (FESEM), raman spectroscopy and fourier transform infrared (FTIR). The properties of the sensor were evaluated by electrochemical techniques. Under the optimal conditions, the peak currents of L-5-HIP were found to be linear in the concentration range of 0.05 μM–7.0 μM, while the sensor also exhibited great features such as low detection limit of 6.0 nM (S/N = 3), superb selectivity against the structural analogues, good antidisturbance ability among coexisting components, excellent repeatability and stability. Moreover, the proposed method had been applied to the detection of L-5-HTP in human blood serum with a satisfactory recoveries ranging from 90.6% to 105.6%.

      PubDate: 2017-03-22T08:29:39Z
      DOI: 10.1016/j.ab.2017.03.017
  • Sulfatase activity assay using an activity-based probe by generation of
           N-methyl isoindole under reducing conditions
    • Authors: Hey Young Yoon; Jong-In Hong
      Abstract: Publication date: Available online 16 March 2017
      Source:Analytical Biochemistry
      Author(s): Hey Young Yoon, Jong-In Hong
      Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. This is an important step in several biological processes such as cellular degradation, hormone regulation, and cell signaling. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions, containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), exhibiting little change in kinetic parameters compared to non-reducing conditions. Probe 1 displayed reasonable kinetic parameters under both non-reducing and reducing conditions, among which the use of Tris buffer and Tris buffer containing GSH appeared to be appropriate conditions for inhibitor screening. Probe 1 showed stronger intensity upon treatment with sulfatase under neutral conditions than under acidic conditions, but it still has limitations in the selectivity for a specific sulfatase. Nevertheless, the fluorescent signal generated as a result of the release of N-methylisoindole after treatment of probe 1 with sulfatase provides a new assay for measuring sulfatase activity that could be adapted for high throughput screening.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.03.012
  • Development of a liquid chromatography-mass spectrometry based enzyme
           activity assay for phosphatidylcholine-specific phospholipase C
    • Authors: Chiaki Murakami; Satoru Mizuno; Sayaka Kado; Fumio Sakane
      Abstract: Publication date: Available online 14 March 2017
      Source:Analytical Biochemistry
      Author(s): Chiaki Murakami, Satoru Mizuno, Sayaka Kado, Fumio Sakane
      Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.03.010
  • Label-free and rapid detection of ATP based on structure switching of
    • Authors: Danyang Ji; Hongqi Wang; Jia Ge; Lin Zhang; Jianjun Li; Dongmei Bai; Juan Chen; Zhaohui Li
      Abstract: Publication date: Available online 14 March 2017
      Source:Analytical Biochemistry
      Author(s): Danyang Ji, Hongqi Wang, Jia Ge, Lin Zhang, Jianjun Li, Dongmei Bai, Juan Chen, Zhaohui Li
      In this work, an aptamer-based fluorescent strategy for label-free detection of ATP was developed by using Thioflavin T (ThT) as a fluorescence indicator, which can specifically bind with G-quadruplex DNAs to generate enhanced fluorescence intensity. In the absence of ATP, the folded structure of ATP aptamer allows the intercalation of ThT to produce strong fluorescence signal. However, upon ATP binding to the aptamer where ThT intercalated, the conformational change or distortion of the aptamer is large enough to cause much less intercalation of ThT and consequently drastic suppression of the fluorescence intensity. As such, the concentration of ATP could be identified very easily by observing fluorescence changes of this sensing system. This label-free assay could be accomplished very easily and quickly with a “mix-and-detect” detection method and exhibits high sensitivity to ATP with a detection limit of 33 nM in a wide range of 0.1–1000 μM. Furthermore, this proposed method is capable of detecting ATP in human serum and cell extracts. This method offers several advantages such as simplicity, rapidity, low cost, good stability and excellent selectivity, which make it hold great potential for the detection of ATP in bioanalytical and biological studies.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.03.011
  • An effective and efficient method of transfecting primary human
           chondrocytes in suspension
    • Authors: Mohammad Shahidul Makki; Nahid Akhtar; Tariq M. Haqqi
      Abstract: Publication date: Available online 14 March 2017
      Source:Analytical Biochemistry
      Author(s): Mohammad Shahidul Makki, Nahid Akhtar, Tariq M. Haqqi
      Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80–90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.

      PubDate: 2017-03-17T04:33:20Z
      DOI: 10.1016/j.ab.2017.03.009
  • Spectrophotometric method for the quantitative assay of
           N-hydroxysulfosuccinimide esters including extinction coefficients and
           reaction kinetics
    • Authors: Rivo Presentini
      Abstract: Publication date: Available online 6 March 2017
      Source:Analytical Biochemistry
      Author(s): Rivo Presentini
      A quantitative spectrophotometric method has been developed for the analysis of N-hydroxysulfosuccinimide (sulfo-NHS), a chromophore with a maximum absorbance at 268 nm. The extinction coefficients were determined between pH 6.0 and 8.0 and found to vary in a nonlinear manner. This spectrophotometric profile is not present in its esters which however release an equimolar amount of sulfo-NHS when they react with nucleophilic groups or hydrolyze in aqueous solution. This fact facilitates the determination in solution of the concentration and purity of bis(sulfosuccinimidyl) suberate (BS3) used as a model, as well as the examination of hydrolysis and aminolysis half-lives in different reaction conditions, these parameters being valuable in optimization of the use of the active esters.

      PubDate: 2017-03-09T20:39:51Z
      DOI: 10.1016/j.ab.2017.03.002
  • Development of a paper-based microfluidic analytical device by a more
           facile hydrophobic substrate generation strategy
    • Authors: Yuan-Yuan Xue; Wen-Tao Zhang; Meng-Yue Zhang; Li-Zhi Liu; Wen-Xin Zhu; Ling-Zhi Yan; Jing Wang; Yan-Ru Wang; Jian-Long Wang; Dao-Hong Zhang
      Abstract: Publication date: Available online 3 March 2017
      Source:Analytical Biochemistry
      Author(s): Yuan-Yuan Xue, Wen-Tao Zhang, Meng-Yue Zhang, Li-Zhi Liu, Wen-Xin Zhu, Ling-Zhi Yan, Jing Wang, Yan-Ru Wang, Jian-Long Wang, Dao-Hong Zhang
      Microfluidic paper-based analytical devices (μPADs) have a significant potential in developing portable and disposable point-of-care testing (POCT). Herein, a facile, rapid, cost-effective and environment friendly strategy for μPADs fabrication is proposed. Specifically, the substrate paper was hydrophobized by coating with trimethoxysilane (TOS), and then the selected area was hydrophilized by treating with surfactant. The whole fabrication process was implemented within 7 min, with no need for complex pre-treatment, high-temperature and special equipment. As a proof-of-concept application, the as-prepared μPAD was applied to determination of the glucose content in human serum samples. The results agreed well with those obtained by a glucometer. We believe that the μPADs fabrication method proposed here could provide a facile, rapid and low-cost reference for other related studies.

      PubDate: 2017-03-03T20:34:58Z
      DOI: 10.1016/j.ab.2017.03.001
  • Engineering a switch-based biosensor for arginine using a Thermotoga
           maritima periplasmic binding protein
    • Authors: Teraya Donaldson; Luisa Iozzino; Lindsay J. Deacon; Hilbert Billones; Alessio Ausili; Sabato D'Auria; Jonathan D. Dattelbaum
      Abstract: Publication date: Available online 1 March 2017
      Source:Analytical Biochemistry
      Author(s): Teraya Donaldson, Luisa Iozzino, Lindsay J. Deacon, Hilbert Billones, Alessio Ausili, Sabato D'Auria, Jonathan D. Dattelbaum
      The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 μM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.

      PubDate: 2017-03-03T20:34:58Z
      DOI: 10.1016/j.ab.2017.02.021
  • Kinetics and inhibition study of tyrosinase by pressure mediated
    • Authors: Dong-Mei Liu; Jun-Li Yang; Wei Ha; Juan Chen; Yan-Ping Shi
      Abstract: Publication date: Available online 1 March 2017
      Source:Analytical Biochemistry
      Author(s): Dong-Mei Liu, Jun-Li Yang, Wei Ha, Juan Chen, Yan-Ping Shi
      In the present study, pressure mediated microanalysis (PMMA), a fast, convenient and efficient capillary electrophoresis (CE) method was developed for studying enzyme kinetics of tyrosinase and inhibition kinetics of kojic acid, a model inhibitor of tyrosinase. The enzymatic reaction conditions and CE conditions were optimized in order to obtain high enzyme activity and short analysis time. By PMMA, only the product could be detected at 475 nm, and no voltage was applied to separate the product from the reaction mixture thus greatly simplifying the optimization procedure. The spectrophotometric assay and electrophoretically mediated microanalysis (EMMA) were also performed to validate the developed method. With the present method, the Michaelis-Menten constant (K m) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (K I) and kojic acid to tyrosinase/L-DOPA complex (K IS) were calculated to be 36.64 and 74.35 μM, respectively, and the half-maximal inhibitory concentration (IC50) was determined to be 46.64 μM for kojic acid. The developed method is fast and convenient for studying enzyme kinetics, inhibition kinetics and further screening enzyme inhibitors.

      PubDate: 2017-03-03T20:34:58Z
      DOI: 10.1016/j.ab.2017.02.020
  • Engineering an FMN-based iLOV protein for the detection of arsenic ions
    • Authors: Yuvaraj Ravikumar; Saravanan prabhu Nadarajan; Chong-soon Lee; Hyungdon Yun
      Abstract: Publication date: Available online 27 February 2017
      Source:Analytical Biochemistry
      Author(s): Yuvaraj Ravikumar, Saravanan prabhu Nadarajan, Chong-soon Lee, Hyungdon Yun
      Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As3+ ions. iLOVN468S exhibited an improved binding affinity with a dissociation constant of 1.5 μM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach.

      PubDate: 2017-03-03T20:34:58Z
      DOI: 10.1016/j.ab.2017.02.012
  • High throughput selection of antibiotic-resistant transgenic Arabidopsis
    • Authors: Yukihiro Nagashima; Hisashi Koiwa
      Abstract: Publication date: Available online 27 February 2017
      Source:Analytical Biochemistry
      Author(s): Yukihiro Nagashima, Hisashi Koiwa
      Kanamycin-resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.

      PubDate: 2017-03-03T20:34:58Z
      DOI: 10.1016/j.ab.2017.02.017
  • In-situ protein determination to monitor contamination in a centrifugal
           partition chromatograph
    • Authors: Feriel Bouiche; Karine Faure
      Abstract: Publication date: Available online 24 February 2017
      Source:Analytical Biochemistry
      Author(s): Feriel Bouiche, Karine Faure
      Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N+ groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes.

      PubDate: 2017-02-24T20:29:33Z
      DOI: 10.1016/j.ab.2017.02.015
  • The feature selection approach for evaluation of potential rheumatoid
           arthritis markers using MALDI-TOF datasets
    • Authors: Bartosz Urbaniak; Piotr Nowicki Dorota Sikorska Samborski Zenon Kokot
      Abstract: Publication date: Available online 24 February 2017
      Source:Analytical Biochemistry
      Author(s): Bartosz Urbaniak, Piotr Nowicki, Dorota Sikorska, Włodzimierz Samborski, Zenon J. Kokot
      Background The selection of the most representative mass profiles, in rheumatoid arthritis (RA) serum samples was developed. This allows for selection and identification of potential biomarkers in RA serum samples. Methods The RA and controls samples were analyzed using MALDI-TOF. Two different protein elution procedures utilizing ZipTips (E1 and E2) were examined. The statistical evaluation of data was performed using different feature selection (FS) methods in combination with different classifiers, while identification of selected masses was performed using MALDI-TOF-TOF. Results Utilization of proposed statistical strategy allowed for the selection of different masses according to FS method and elution procedure. Obtained masses were further subjected for targeted identification. The panel of proteins were identified as potential markers. The role of these proteins was discussed in relation to pathomechanism of RA. Conclusion Application of advanced biostatistical analysis of obtained MALDI-TOF datasets, resulted with targeted selection of potential RA biomarkers. Five proteins were identified due the E1 procedure, and six proteins were identified due the E2 procedure, respectively. The panel of identified proteins suggest that presented statistical methodology and proteomic strategy was correct and gave valid results. Obtained results may contribute to development of clinically useful multicomponent diagnostic tool.

      PubDate: 2017-02-24T20:29:33Z
  • Identification and characterization of glycation adducts on osteocalcin
    • Authors: Corinne J. Thomas; Timothy P. Cleland; Sheng Zhang; Caren M. Gundberg; Deepak Vashishth
      Abstract: Publication date: Available online 22 February 2017
      Source:Analytical Biochemistry
      Author(s): Corinne J. Thomas, Timothy P. Cleland, Sheng Zhang, Caren M. Gundberg, Deepak Vashishth
      Osteocalcin is an important extracellular matrix bone protein that contributes to the structural properties of bone through its interactions with hydroxyapatite mineral and with collagen I. This role may be affected by glycation, a labile modification the levels of which has been shown to correlate with bone fragility. Glycation starts with the spontaneous addition of a sugar onto a free amine group on a protein, forming an Amadori product, and then proceeds through several environment-dependent stages resulting in the formation of an advanced glycation end product. Here, we induce the first step of this modification on synthetic osteocalcin, and then use multiple mass spectrometry fragmentation techniques to determine the location of this modification. Collision-induced dissociation resulted in spectra dominated by neutral loss, and was unable to identify Amadori products. Electron-transfer dissociation showed that the Amadori product formed solely on osteocalcin's N-terminus. This suggests that the glycation of osteocalcin is unlikely to interfere with osteocalcin's interaction with hydroxyapatite. Instead, glycation may interfere with its interaction with collagen I or another bone protein, osteopontin. Potentially, the levels of glycated osteocalcin fragments released from bone during bone resorption could be used to assess bone quality, should the N-terminal fragments be targeted.

      PubDate: 2017-02-24T20:29:33Z
      DOI: 10.1016/j.ab.2017.02.011
  • Synthesis of hydroxyethyl-methacrylate-(L)-histidine methyl ester
           cryogels. Application on the separation of bovine immunoglobulin G
    • Authors: Assem Elkak; Amar Hamade; Nilay Bereli; Canan Armutcu; Adil Denizli
      Abstract: Publication date: Available online 21 February 2017
      Source:Analytical Biochemistry
      Author(s): Assem Elkak, Amar Hamade, Nilay Bereli, Canan Armutcu, Adil Denizli
      In this study cryogels based 2-hydroxyethyl methacrylate (HEMA) functionalized with N-methacryloyl-L-histidine methyl ester (MAH) were synthesized and used for the adsorption and separation of bovine IgG. Two series of cryogels functionalized with 5 and 10 mg of MAH as pseudobioaffinity ligand were prepared and characterized by swelling test, FTIR and SEM analysis. The adsorption efficiency of the bovine immunoglobulin into cryogels is discussed with respect to the following chromatographic parameters: pH, flow rate, initial IgG concentration, adsorption time and ionic strength. Our results show good adsorption of bovine immunoglobulin under mild separation conditions at pH 7.4. The maximum binding capacity was determined (32.4 mg/g of cryogel) and demonstrates the efficiency of the used cryogels. This efficacy is clearly seen upon increasing the maximum binding capacity from 23.2 mg (obtained with cryogels with 5 mg MAH) to 32.4 mg/g (for cryogel with 10 mg MAH ligand concentration). The purity of separated fractions was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Together our observations highlights poly (HEMA-MAH) as an efficient adsorbent for bovine immunoglobulins G separation.

      PubDate: 2017-02-24T20:29:33Z
      DOI: 10.1016/j.ab.2017.02.003
  • Detection of histidine-rich glycoprotein and fibrinogen with nickel-enzyme
           conjugates: Purification of rabbit HRG
    • Authors: Mathia Colwell; Najma Ahmed; Ralph Butkowski
      Abstract: Publication date: Available online 21 February 2017
      Source:Analytical Biochemistry
      Author(s): Mathia Colwell, Najma Ahmed, Ralph Butkowski
      Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG)1 and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG.

      PubDate: 2017-02-24T20:29:33Z
      DOI: 10.1016/j.ab.2017.02.013
  • Exploring sensitivity & throughput of a parallel flow SPRi biosensor
           for characterization of antibody-antigen interaction
    • Authors: Vishal Kamat; Ashique Rafique
      Abstract: Publication date: Available online 20 February 2017
      Source:Analytical Biochemistry
      Author(s): Vishal Kamat, Ashique Rafique
      Rapid growth in the field of biotherapeutics has led to an increased demand for high-throughput, label-free biosensors exhibiting high sensitivity. To support the current needs, Sierra Sensors introduced a surface plasmon resonance imaging (SPRi) based biosensor, Molecular Affinity Screening System (MASS-1). We assessed the potential utility of MASS-1 to support Regeneron's therapeutic antibody discovery. A large panel of antibody-antigen interactions was characterized using MASS-1 and the kinetic data were compared with the Biacore 4000 biosensor. Less than 10% deviation in the binding rate constants measured across eight flow channels of MASS-1 was observed. The single injection cycle kinetic assay allowed rapid measurement of binding rate constants for antibody-antigen interactions. MASS-1 sensitivity is independent of protein immobilization level and kinetic analysis performed using ultra-low density mAb surfaces allowed characterization of picomolar affinity interactions without mass transport limitation. High-throughput characterization of a panel of 189 monoclonal antibodies to 13 different antigens with molecular weights ranging from 14kD to 105kD revealed that binding kinetic parameters measured on MASS-1 were comparable to those measured on Biacore 4000. Our data demonstrate that MASS-1 measures reliable binding kinetic parameters and has an appropriate combination of throughput and sensitivity to support discovery and development of therapeutic antibodies.

      PubDate: 2017-02-24T20:29:33Z
      DOI: 10.1016/j.ab.2017.02.007
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