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Publisher: Elsevier   (Total: 3042 journals)

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Showing 1 - 200 of 3042 Journals sorted alphabetically
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 19, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 16, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 81, SJR: 1.109, h-index: 94)
Accounting Forum     Hybrid Journal   (Followers: 23, 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: 328, 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: 205, 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: 22, 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: 4, 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: 3)
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: 124, 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: 9, 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: 25, 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: 25, 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: 40, 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: 45, 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: 12)
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: 20, 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: 24)
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: 34, 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: 8, SJR: 0.764, h-index: 15)
Advances in Lipobiology     Full-text available via subscription   (Followers: 2)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 9)
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: 22)
Advances in Molecular and Cellular Endocrinology     Full-text available via subscription   (Followers: 10)
Advances in Molecular Toxicology     Full-text available via subscription   (Followers: 7, SJR: 0.324, h-index: 8)
Advances in Nanoporous Materials     Full-text available via subscription   (Followers: 4)
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: 21, SJR: 0.4, h-index: 28)
Advances in Pharmaceutical Sciences     Full-text available via subscription   (Followers: 13)
Advances in Pharmacology     Full-text available via subscription   (Followers: 15, 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: 19, SJR: 1.5, h-index: 62)
Advances in Psychology     Full-text available via subscription   (Followers: 58)
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: 2, SJR: 0.1, h-index: 2)
Advances in Space Research     Full-text available via subscription   (Followers: 339, SJR: 0.606, h-index: 65)
Advances in Structural Biology     Full-text available via subscription   (Followers: 8)
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: 29, SJR: 1.321, h-index: 56)
Advances in Veterinary Medicine     Full-text available via subscription   (Followers: 15)
Advances in Veterinary Science and Comparative Medicine     Full-text available via subscription   (Followers: 13)
Advances in Virus Research     Full-text available via subscription   (Followers: 5, SJR: 1.878, h-index: 68)
Advances in Water Resources     Hybrid Journal   (Followers: 43, 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: 308, 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: 5, 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: 422, 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: 30, SJR: 1.275, h-index: 74)
Agricultural Water Management     Hybrid Journal   (Followers: 38, 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: 50, 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: 5, 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: 10, SJR: 0.922, h-index: 66)
Alcoholism and Drug Addiction     Open Access   (Followers: 6)
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: 8, 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: 46, 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: 47, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 44, 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: 15, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 30, SJR: 8.769, h-index: 256)
American J. of Infection Control     Hybrid Journal   (Followers: 24, SJR: 1.259, h-index: 81)
American J. of Kidney Diseases     Hybrid Journal   (Followers: 32, SJR: 2.313, h-index: 172)
American J. of Medicine     Hybrid Journal   (Followers: 44, 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: 179, SJR: 2.255, h-index: 171)
American J. of Ophthalmology     Hybrid Journal   (Followers: 54, 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: 23, 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: 33, SJR: 1.286, h-index: 125)
American J. of the Medical Sciences     Hybrid Journal   (Followers: 12, 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: 53, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 5)
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: 160, SJR: 0.725, h-index: 154)
Analytical Chemistry Research     Open Access   (Followers: 8, 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: 153, 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]   [160 followers]  Follow
    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3042 journals]
  • A refined DNA methylation detection method using MspJI coupled
           quantitative PCR
    • Authors: Christopher J. Petell; Gilbert Loiseau; Ryan Gandy; Sriharsa Pradhan; Humaira Gowher
      Pages: 1 - 9
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Christopher J. Petell, Gilbert Loiseau, Ryan Gandy, Sriharsa Pradhan, Humaira Gowher
      DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.

      PubDate: 2017-06-22T03:42:09Z
      DOI: 10.1016/j.ab.2017.06.006
      Issue No: Vol. 533 (2017)
       
  • Selective fluorescence detection method for selenide and selenol using
           monochlorobimane
    • Authors: Takeshi Imai; Tatsuo Kurihara; Nobuyoshi Esaki; Hisaaki Mihara
      Pages: 1 - 8
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Takeshi Imai, Tatsuo Kurihara, Nobuyoshi Esaki, Hisaaki Mihara
      The low redox potential of selenide and selenol is physiologically important, as it confers efficient catalytic abilities to selenoproteins. Quantitative determination of selenol and selenide provide important clues for understanding the metabolism and physiological function of selenium. However, selective detection of selenol and selenide is extremely difficult because of their chemical similarity to thiol and sulfide. In this study, we established a highly sensitive, selective, quantitative, and simple method for detection of selenol and selenide, using a reaction with monochlorobimane (MCB), followed by ethyl acetate extraction of the product syn-(methyl,methyl)bimane. We analyzed selenide production from selenite, catalyzed by human glutathione reductase, and also determined selenide and selenol concentrations in Hepa1-6 cells using the MCB method, to demonstrate its practical applications. This study provides a new tool for selenium detection in biology.

      PubDate: 2017-06-06T12:06:07Z
      DOI: 10.1016/j.ab.2017.05.023
      Issue No: Vol. 532 (2017)
       
  • Detection and purification of backbone-cyclized proteins using a
           bacterially expressed anti-myc-tag single chain antibody
    • Authors: Sushma Chauhan; Chen Yuan Hou; Sang Taek Jung; Taek Jin Kang
      Pages: 38 - 44
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Sushma Chauhan, Chen Yuan Hou, Sang Taek Jung, Taek Jin Kang
      A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2017.06.003
      Issue No: Vol. 532 (2017)
       
  • Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay
           with a chromophoric substrate
    • Authors: Nicole M. Luzi; Charles E. Lyons; Darrell L. Peterson; Keith C. Ellis
      Pages: 45 - 52
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Nicole M. Luzi, Charles E. Lyons, Darrell L. Peterson, Keith C. Ellis
      Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKACα inhibitors H89 and PKI(5-24). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-32P-ATP. The assay format is also highly flexible and could be adapted for the testing of many different kinases by changing the peptide substrate sequence.

      PubDate: 2017-06-16T14:27:14Z
      DOI: 10.1016/j.ab.2017.06.001
      Issue No: Vol. 532 (2017)
       
  • Effective cell-free drug screening protocol for protein-protein
           interaction
    • Authors: Shaked Ashkenazi; Alexander Plotnikov; Anat Bahat; Rivka Dikstein
      Pages: 53 - 59
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Shaked Ashkenazi, Alexander Plotnikov, Anat Bahat, Rivka Dikstein
      Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI. This enables production of large quantities of the RL fusion proteins in a simple and cost effective manner that is suitable for very large screens. Subsequently, inhibitory compounds are analyzed in a similar complementation assay in living cultured mammalian cells to select for those that can penetrate cells. We applied this method to NF-κB, a family of dimeric transcription factors that plays central roles in immune responses, cell survival and aging, and its dysregulation is linked to many pathological states. This strategy led to the identification of several direct NF-κB inhibitors. As the described protocol is very straightforward and robust it may be suitable for many pairs of interacting proteins.

      PubDate: 2017-06-16T14:27:14Z
      DOI: 10.1016/j.ab.2017.05.030
      Issue No: Vol. 532 (2017)
       
  • Single molecule Raman spectroscopic assay to detect transgene from GM
           plants
    • Authors: Ulhas S. Kadam; Rahul L. Chavhan; Burkhard Schulz; Joseph Irudayaraj
      Pages: 60 - 63
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Ulhas S. Kadam, Rahul L. Chavhan, Burkhard Schulz, Joseph Irudayaraj
      Substantial concerns have been raised for the safety of transgenics on human health and environment. Many organizations, consumer groups, and environmental agencies advocate for stringent regulations to avoid transgene products' contamination in food cycle or in nature. Here we demonstrate a novel approach using surface enhanced Raman spectroscopy (SERS) to detect and quantify transgene from GM plants. We show a highly sensitive and accurate quantification of transgene DNA from multiple transgenic lines of Arabidopsis. The assay allows us to detect and quantify the transgenes as low as 0.10 pg without need for PCR-amplification. This technology is relatively cheap, quick, simple, and suitable for detection at low target concentration.

      PubDate: 2017-06-16T14:27:14Z
      DOI: 10.1016/j.ab.2017.06.002
      Issue No: Vol. 532 (2017)
       
  • Electrochemical label-free and sensitive nanobiosensing of DNA
           hybridization by graphene oxide modified pencil graphite electrode
    • Authors: F. Ahour; A. Shamsi
      Pages: 64 - 71
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): F. Ahour, A. Shamsi
      Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π– π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM–0.5 μM with a detection limit of 4.3 × 10−11 M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future.

      PubDate: 2017-06-22T03:42:09Z
      DOI: 10.1016/j.ab.2017.06.004
      Issue No: Vol. 532 (2017)
       
  • Photometric assay of maltose and maltose-forming enzyme activity by using
           4-alpha-glucanotransferase (DPE2) from higher plants
    • Authors: Julia Smirnova; Alisdair R. Fernie; Christian C.M. Spahn; Martin Steup
      Pages: 72 - 82
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Julia Smirnova, Alisdair R. Fernie, Christian C.M. Spahn, Martin Steup
      Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify β-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels.

      PubDate: 2017-06-22T03:42:09Z
      DOI: 10.1016/j.ab.2017.05.026
      Issue No: Vol. 532 (2017)
       
  • Thioflavin T fluorescence to analyse amyloid formation kinetics:
           Measurement frequency as a factor explaining irreproducibility
    • Authors: Mathew Sebastiao; Noe Quittot; Steve Bourgault
      Pages: 83 - 86
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Mathew Sebastiao, Noe Quittot, Steve Bourgault
      The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence. This effect, attributed to agitation during microplate displacements between fluorescence measurements, reinforces the importance of a better standardization in amyloid formation assays.

      PubDate: 2017-06-22T03:42:09Z
      DOI: 10.1016/j.ab.2017.06.007
      Issue No: Vol. 532 (2017)
       
  • Dopamine modified hyperbranched TiO2 arrays based ultrasensitive
           photoelectrochemical immunosensor for detecting neuron specific enolase
    • Authors: He Li; Qiyou Xiao; Jiaxin Lv; Qin Lei; Yujie Huang
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): He Li, Qiyou Xiao, Jiaxin Lv, Qin Lei, Yujie Huang
      In this work, three-dimensional (3D) hyperbranched TiO2 nanorod arrays were synthesized and used to fabricate dopamine sensitized photoelectrochemical (PEC) biosensor. To increase the lifetime of charge carriers and enhance the photocurrent responses signal, a delicate signal amplification strategy by introducing dopamine (DA) as sensitizer was developed. The dopamine sensitized TiO2 can shorten the carrier diffusion distance, improve light harvesting efficiency and charge collection efficiency, which results in performance improvement of the as-obtained PEC sensor. This proposed biosensor for determination of neuron specific enolase (NSE) demonstrated a good linear relationship range from 0.1 ng mL−1 to 1000 ng mL−1 with a detection limit of 0.05 ngmL−1 (S/N = 3). In addition, the as-prepared immunosensor exhibits excellent selectivity, stability and reproducibility, which could be extended to other label-free sensing fields. Therefore, this proposed method may also provide potential applications for the clinical examination.

      PubDate: 2017-05-27T13:26:21Z
      DOI: 10.1016/j.ab.2017.05.025
      Issue No: Vol. 531 (2017)
       
  • Development of a ssDNA aptamer for detection of residual benzylpenicillin
    • Authors: A-Young Lee; Na-Reum Ha; In-Pil Jung; Sang-Heon Kim; A-Ru Kim; Moon-Young Yoon
      Pages: 1 - 7
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): A-Young Lee, Na-Reum Ha, In-Pil Jung, Sang-Heon Kim, A-Ru Kim, Moon-Young Yoon
      Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, Kd = 383.4 nM, and had a low limit of detection (LOD) of 9.2 nM. The characterization of the aptamer was performed through the fluorescence recovery signal from rGO surface. In addition, detection of benzylpenicillin was performed in pretreated milk samples, and its detection accuracy was shown to be 100 ± 10%. This represented that BBA1 was used for fluorescence aptasensor system in real sample. Furthermore, this benzylpenicillin binding aptamer showed high specificity against other antibiotics except for ampicillin. With these advantageous characteristics, we expect that this aptamer could be applied to an on-site detection system for residual benzylpenicillin.

      PubDate: 2017-05-22T13:15:54Z
      DOI: 10.1016/j.ab.2017.05.013
      Issue No: Vol. 531 (2017)
       
  • Aptatope mapping of the binding site of a progesterone aptamer on the
           steroid ring structure
    • Authors: Vasso Skouridou; Thomas Schubert; Abdulaziz S. Bashammakh; Mohammad S. El-Shahawi; Abdulrahman O. Alyoubi; Ciara K. O'Sullivan
      Pages: 8 - 11
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Vasso Skouridou, Thomas Schubert, Abdulaziz S. Bashammakh, Mohammad S. El-Shahawi, Abdulrahman O. Alyoubi, Ciara K. O'Sullivan
      In this work we report the mapping of the binding site of the only progesterone aptamer published to date, in an approach referred to as aptatope mapping. By linking the binding data obtained from microscale thermophoresis analysis to the structural differences on the ring structure of a range of steroids, we elucidated the moieties involved in aptamer-progesterone binding. This approach can be further exploited for the characterization of aptamer specificity and ultimately facilitate the development of aptamer-based assays depending on the desired specificity.

      PubDate: 2017-05-22T13:15:54Z
      DOI: 10.1016/j.ab.2017.05.010
      Issue No: Vol. 531 (2017)
       
  • Solid-phase synthesis of highly repetitive chromatin assembly templates
    • Authors: Melissa J. Blacketer; Margaret K. Gannon; Isaac A. Young; Michael A. Shogren-Knaak
      Pages: 12 - 15
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Melissa J. Blacketer, Margaret K. Gannon, Isaac A. Young, Michael A. Shogren-Knaak
      DNA templates for assembling chromatin model systems typically consist of numerous repeats of nucleosome positioning sequences, making their synthesis challenging. Here we describe a solid-phase strategy for generating such templates using sequential enzymatic ligation of DNA monomers. Using single nucleosome site monomers, we can either generate a twelve-nucleosome site target, or systematically access intermediate-sized templates. Using twelve nucleosome positioning site monomers, longer templates can be generated. Our synthesized templates assemble into well-defined chromatin model systems, demonstrating the utility of our solid-phase approach. Moreover, our strategy should be more widely applicable to generating other DNAs containing highly repetitive DNA sequences.

      PubDate: 2017-05-22T13:15:54Z
      DOI: 10.1016/j.ab.2017.05.007
      Issue No: Vol. 531 (2017)
       
  • An optical alignment system improves precision of soluble aggregate
           quantitation by sedimentation velocity analytical ultracentrifugation
    • Authors: Brandon L. Doyle; Ivan L. Budyak; Adam P. Rauk; William F. Weiss
      Pages: 16 - 19
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Brandon L. Doyle, Ivan L. Budyak, Adam P. Rauk, William F. Weiss
      Appropriate characterization of soluble aggregates is an important aspect of biologics development and manufacturing, and sedimentation velocity analytical ultracentrifugation (SV-AUC) is often used an orthogonal technique to size-exclusion chromatography (SEC) for this purpose. Precise quantification of low levels of soluble aggregates by SV-AUC can be adversely impacted by improper cell alignment. This report describes the development of an optical system capable of quantifying cell alignment that affords a substantial improvement compared to historical approaches.

      PubDate: 2017-05-27T13:26:21Z
      DOI: 10.1016/j.ab.2017.05.018
      Issue No: Vol. 531 (2017)
       
  • Radiative decay engineering 8: Coupled emission microscopy for lens-free
           high-throughput fluorescence detection
    • Authors: Liangfu Zhu; Ramachandram Badugu; Douguo Zhang; Ruxue Wang; Emiliano Descrovi; Joseph R. Lakowicz
      Pages: 20 - 36
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Liangfu Zhu, Ramachandram Badugu, Douguo Zhang, Ruxue Wang, Emiliano Descrovi, Joseph R. Lakowicz
      Fluorescence spectroscopy and imaging are now used throughout the biosciences. Fluorescence microscopes, spectrofluorometers, microwell plate readers and microarray imagers all use multiple optical components to collect, redirect and focus the emission onto single point or array imaging detectors. For almost all biological samples, except those with regular nanoscale features, emission occurs in all directions. With the exception of complex microscope objectives with large collection angles (NA ≤ 0.5), all these instruments collect only a small fraction of the total emission. Because of the increasing knowledge base on fluorophores within near-field (<200 nm) distances from plasmonic and photonic structures we can anticipate the development of compact devices in which the sample to be detected is located directly on solid state detectors such as CCDs or CMOS cameras. Near-field interactions of fluorophores with metallic or dielectric multi-layer structures (MLSs) can capture a large fraction of the total emission. Depending on the composition and dimensions of the MLSs, the spatial distribution of the sample emission results in distinct optical patterns on the detector surface. With either plain glass slides or MLSs the most commonly used front focal plane (FFP) images reveal the x-y spatial distribution of emission from the sample. Another approach, which is often used with two or three-dimensional nanostructures, is back focal plane (BFP) imaging. The BFP images reveal the angular distribution of the emission. The FFP and BFP images occur at certain distances from the sample which is determined by the details of the optical components. Obtaining these images requires multiple optical components and distances which are too large for the compact devices. For devices described in this paper, the images will be detected at a fixed distance between the sample and some arbitrary distance below the MLS which is determined by the geometry and thicknesses of the components. We refer to measurements at these locations as out-of-focal plane (OFP) imaging. Herein we describe a method to measure the optical fields at micron and multi-micron distances below the MLS, which will represent the images seen by an optically coupled array detector. The possibility of sub-surface optical images is illustrated using five different multi-layer structures. This is accomplished using an optical configuration which allows measurement at a front focal plane (FFP), back focal plane (BFP) or any OFP locations. Our OFP imaging method provides a link between the FFP images which reveals the surface distribution of fluorophores with the BFP images that reveal the angular distribution of emission. This linkage can be useful when examining structures which have nanoscale features due to fluorescence or leakage radiation from nanostructures.
      Graphical abstract image

      PubDate: 2017-05-27T13:26:21Z
      DOI: 10.1016/j.ab.2017.05.020
      Issue No: Vol. 531 (2017)
       
  • Ligation-mediated PCR with a back-to-back adapter reduces amplification
           bias resulting from variations in GC content
    • Authors: Satoru Ishihara; Naoe Kotomura; Naoki Yamamoto; Hiroshi Ochiai
      Pages: 37 - 44
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Satoru Ishihara, Naoe Kotomura, Naoki Yamamoto, Hiroshi Ochiai
      Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis.

      PubDate: 2017-05-27T13:26:21Z
      DOI: 10.1016/j.ab.2017.05.011
      Issue No: Vol. 531 (2017)
       
  • Isolation of single cells for protein therapeutics using microwell
           selection and Surface Plasmon Resonance imaging
    • Authors: F. Abali; M. Stevens; A.G.J. Tibbe; L.W.M.M. Terstappen; P.N. van der Velde; R.B.M. Schasfoort
      Pages: 45 - 47
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): F. Abali, M. Stevens, A.G.J. Tibbe, L.W.M.M. Terstappen, P.N. van der Velde, R.B.M. Schasfoort
      Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation.

      PubDate: 2017-05-27T13:26:21Z
      DOI: 10.1016/j.ab.2017.05.021
      Issue No: Vol. 531 (2017)
       
  • Generation of SMURF2 knockout human cells using the CRISPR/Cas9 system
    • Authors: Dhanoop Manikoth Ayyathan; Nataša Ilić; Hava Gil-Henn; Michael Blank
      Pages: 56 - 59
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Dhanoop Manikoth Ayyathan, Nataša Ilić, Hava Gil-Henn, Michael Blank
      The HECT domain E3 ubiquitin ligase SMURF2 regulates stability of several key protein targets involved in tumorigenesis, cell proliferation, migration, differentiation, and senescence. While altered levels and aberrant cellular distribution of SMURF2 were reported in different types of cancer, its role in tumorigenesis is far from understood. To elucidate the role of SMURF2 in cancer, appropriate human cancer cell models are needed. Here, we describe approaches that can be used to generate human normal and cancer cell strains knocked-out for SMURF2 using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing technology.

      PubDate: 2017-06-02T11:08:42Z
      DOI: 10.1016/j.ab.2017.05.024
      Issue No: Vol. 531 (2017)
       
  • Simultaneous production of monoclonal antibodies against Bacillus
           thuringiensis (Bt) Cry1 toxins using a mixture immunization
    • Authors: Sa Dong; Cunzheng Zhang; Yuan Liu; Xiao Zhang; Yajing Xie; Jianfeng Zhong; Chongxin Xu; Xianjin Liu
      Pages: 60 - 66
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Sa Dong, Cunzheng Zhang, Yuan Liu, Xiao Zhang, Yajing Xie, Jianfeng Zhong, Chongxin Xu, Xianjin Liu
      The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6.37–11.35 ng mL−1 and 17.36–24.48 ng mL−1, respectively. The recovery tests showed that the recoveries ranged from 78% to 110% within the quantitation range (LOQ-100 ng mL−1). The established DAS-ELISA can be a useful tool for monitoring the Cry1 toxins in agricultural products. Mixture immunization opens a new path for producing diverse mAbs simultaneously in a single immunization circle.

      PubDate: 2017-06-02T11:08:42Z
      DOI: 10.1016/j.ab.2017.05.016
      Issue No: Vol. 531 (2017)
       
  • Binding assay for characterization of protein kinase inhibitors possessing
           sub-picomolar to sub-millimolar affinity
    • Authors: Hedi Sinijarv; Shanshan Wu; Taavi Ivan; Tonis Laasfeld; Kaido Viht; Asko Uri
      Pages: 67 - 77
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Hedi Sinijarv, Shanshan Wu, Taavi Ivan, Tonis Laasfeld, Kaido Viht, Asko Uri
      High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest K d value that can be accurately determined with the method is 60 fM. We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of K d determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.
      Graphical abstract image

      PubDate: 2017-06-02T11:08:42Z
      DOI: 10.1016/j.ab.2017.05.017
      Issue No: Vol. 531 (2017)
       
  • The photostability of the commonly used biotin-4-fluorescein probe
    • Authors: Richard A. Haack; Kerry M. Swift; Qiaoqiao Ruan; Richard J. Himmelsbach; Sergey Y. Tetin
      Pages: 78 - 82
      Abstract: Publication date: 15 August 2017
      Source:Analytical Biochemistry, Volume 531
      Author(s): Richard A. Haack, Kerry M. Swift, Qiaoqiao Ruan, Richard J. Himmelsbach, Sergey Y. Tetin
      Biotin-4-fluorescein (B4F) is a commonly used fluorescent probe for studying biotin-(strept)avidin interactions. During a characterization study of an anti-biotin antibody, using B4F as the probe, we noticed a discrepancy in the expected and experimentally determined number of biotin binding sites. Analytical testing showed that the biotin moiety in the probe undergoes a photosensitized oxidation to produce a mixture of biotin sulfoxides which has the potential to impact the quantitation of binding sites using this fluorescent probe.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2017.05.019
      Issue No: Vol. 531 (2017)
       
  • Accelerated isothermal nucleic acid amplification in betaine-free reaction
    • Authors: Cuiping Ma; Yifan Wang; Pansong Zhang; Chao Shi
      Pages: 1 - 4
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Cuiping Ma, Yifan Wang, Pansong Zhang, Chao Shi
      Betaine was used as a common additive to isothermal nucleic acid amplification reactions because of lowering the melting temperature (Tm) of DNA. Herein, we reported a novel finding that betaine was inhibiting the reaction efficiency of isothermal amplification reactions. In this work, we have verified this finding by classical loop-mediated isothermal amplification that the addition of 0.8 M betaine inhibited the efficiency of reaction dropping to approximately 1%. Additionally, we clarified the mechanism of betaine hindering isothermal amplification reactions with a molecular barrier to lower associate rate constant K1 for intermolecular hybridization. This finding would be very significant for studies on the interaction between small molecule substance and DNA, and the development of point-of-care testing because of simplifying reaction system and increasing reaction efficiency.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.04.017
      Issue No: Vol. 530 (2017)
       
  • Sequence-independent cloning methods for long DNA fragments applied to
           synthetic biology
    • Authors: N. Dalonso; M. Savoldi; P.H.C. França; T.F. Reis; G.H. Goldman; R.M.M. Gern
      Pages: 5 - 8
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): N. Dalonso, M. Savoldi, P.H.C. França, T.F. Reis, G.H. Goldman, R.M.M. Gern
      Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.04.018
      Issue No: Vol. 530 (2017)
       
  • Deproteinization assessment using isotopically enriched compounds to trace
           the coprecipitation of low-molecular-weight selenium species with proteins
           
    • Authors: Simon Godin; Diego Bouzas-Ramos; Stéphanie Fontagné-Dicharry; Brice Bouyssière; Maïté Bueno
      Pages: 9 - 16
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Simon Godin, Diego Bouzas-Ramos, Stéphanie Fontagné-Dicharry, Brice Bouyssière, Maïté Bueno
      Studies have shown that information related to the presence of low-molecular-weight metabolites is frequently lost after deproteinization of complex matrices, such as blood and plasma, during sample preparation. Therefore, the effect of several deproteinization reagents on low-molecular-weight selenium species has been compared by species-specific isotope labeling. Two isotopically enriched selenium tracers were used to mimic models of small inorganic anionic (77Se-selenite) and organic zwitterionic (76Se-selenomethionine) species. The results presented here show that the use of a methanol-acetonitrile-acetone (1:1:1 v/v/v) mixture provided approximately two times less tracer loss from plasma samples in comparison with the classic procedure using acetonitrile, which may not be optimal as it leads to important losses of low-molecular-weight selenium species. In addition, the possible interactions between selenium tracers and proteins were investigated, revealing that both coprecipitation phenomena and association with proteins were potentially responsible for selenite tracer losses during protein precipitation in blood samples. However, coprecipitation phenomena were found to be fully responsible for losses of both tracers observed in plasma samples and of the selenomethionine tracer in blood samples. This successfully applied strategy is anticipated to be useful for more extensive future studies in selenometabolomics.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.05.002
      Issue No: Vol. 530 (2017)
       
  • Heparan sulfate disaccharide measurement from biological samples using
           pre-column derivatization, UPLC-MS and single ion monitoring
    • Authors: Imeobong U. Antia; Darshna R. Yagnik; Leonardo Pantoja Munoz; Ajit J. Shah; Frank A. Hills
      Pages: 17 - 30
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Imeobong U. Antia, Darshna R. Yagnik, Leonardo Pantoja Munoz, Ajit J. Shah, Frank A. Hills
      Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02–0.15 and 0.07–0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.04.019
      Issue No: Vol. 530 (2017)
       
  • Newborn screening by matrix-assisted laser desorption/ionization mass
           spectrometry based on parylene-matrix chip
    • Authors: Jo-Il Kim; Joo-Yoon Noh; Mira Kim; Jong-Min Park; Hyun-Woo Song; Min-Jung Kang; Jae-Chul Pyun
      Pages: 31 - 39
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Jo-Il Kim, Joo-Yoon Noh, Mira Kim, Jong-Min Park, Hyun-Woo Song, Min-Jung Kang, Jae-Chul Pyun
      Newborn screening for diagnosis of phenylketonuria, homocystinuria, and maple syrup urine disease have been conducted by analyzing the concentration of target amino acids using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) based on parylene-matrix chip. Parylene-matrix chip was applied to MALDI-ToF MS analysis reducing the matrix peaks significantly at low mass-to-charge ratio range (m/z < 500). Reproducibility of inter-spot and intra-spot analyses of amino acids was less than 10%. Methanol extraction was adopted for simple and rapid sample preparation of serum before mass spectrometric analysis showing 13.3 to 45% of extraction efficiency. Calibration curves for diagnosis of neonatal metabolic disorders were obtained by analyzing methanol-extracted serum spiked with target amino acids using MALDI-ToF MS. They showed good linearity (R2 > 0.98) and the LODs were ranging from 9.0 to 22.9 μg/mL. Effect of proteins in serum was estimated by comparing MALDI-ToF mass spectra of amino acids-spiked serum before and after the methanol extraction. Interference of other amino acids on analysis of target analyte was determined to be insignificant. From these results, MALDI-ToF MS based on parylene-matrix chip could be applicable to medical diagnosis of neonatal metabolic disorders.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.04.021
      Issue No: Vol. 530 (2017)
       
  • A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of
           human genes
    • Authors: Kamlesh Bisht; Sherilyn Grill; Jacqueline Graniel; Jayakrishnan Nandakumar
      Pages: 40 - 49
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Kamlesh Bisht, Sherilyn Grill, Jacqueline Graniel, Jayakrishnan Nandakumar
      CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.05.001
      Issue No: Vol. 530 (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
      Pages: 50 - 56
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      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: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.04.014
      Issue No: Vol. 530 (2017)
       
  • Quantification of autophagy flux using LC3 ELISA
    • Authors: Sung-hee Oh; Yong-bok Choi; June-hyun Kim; Conrad C. Weihl; Jeong-sun Ju
      Pages: 57 - 67
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Sung-hee Oh, Yong-bok Choi, June-hyun Kim, Conrad C. Weihl, Jeong-sun Ju
      Macroautophagy (hereafter referred to as autophagy) is a degradation system that delivers cytoplasmic materials to lysosomes via autophagosomes. Autophagic flux is defined as a measure of autophagic degradation activity. Despite several methods for monitoring autophagic flux being currently utilized, interest in finding a highly accurate, sensitive and well-quantifiable assay is still growing. Therefore, we introduce a new approach analyzing autophagic flux in vitro and in vivo using enzyme-linked immunosorbent assay (ELISA) technique. In order to adapt this assay from LC3-II turnover measured by Western blot in the presence and absence of lysosomal inhibitors, we induced autophagy by starvation or rapamycin and mitophagy (mitochondrial degradation by autophagy) by CCCP in C2C12 myotubes for 8 h and in mice for 48 h with and without Bafilomycin A1 or colchicine treatment, respectively. Following subcellular fractionation of mouse skeletal muscle cells and tissue, cytosolic, membrane, and mitochondrial fractions were analyzed through a sandwich ELISA using two LC3 antibodies, LC3 capture and HRP-conjugated LC3 detection antibodies. Using this ELISA, changes in the membrane-bound or mitochondrion-associated LC3-II levels, and the ratio of the LC3-II from each fraction to LC3-I levels (cytosolic fraction) were evaluated for measuring autophagy and mitophagy flux. This study demonstrates that this ELISA was more sensitive and reliable to measure autophagic/mitophagic flux in both in vitro and in vivo, compared with the most commonly used LC3 turnover assay via Western blot.

      PubDate: 2017-05-08T12:49:55Z
      DOI: 10.1016/j.ab.2017.05.003
      Issue No: Vol. 530 (2017)
       
  • A high sensitive epitope imprinted electrochemical sensor for bovine serum
           albumin based on enzyme amplifying
    • Authors: Meng-Xi Li; Xing-Hua Wang; Lian-Ming Zhang; Xiao-Ping Wei
      Pages: 68 - 74
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Meng-Xi Li, Xing-Hua Wang, Lian-Ming Zhang, Xiao-Ping Wei
      To improve the sensitivity of the molecular imprinting sensor detection of protein, a new strategy based on enzyme amplification was proposed. The determination of bovine serum albumin (BSA) was achieved by using the epitope imprinted techniques coupling with electrochemical measurement method. Nonapeptide, separated from BSA, was selected as a template molecule to prepare the molecularly imprinted polymer (MIP) film, and it could bind with the cavities of the MIP. By the use of epitope imprinted techniques, BSA can be recognized by the MIP via the nonapeptide on the surface of BSA. The synthesized horseradish peroxidase-labeled nonapeptide (HRP-nonapeptide) can also be recognized by the MIP. After the competitive reaction between HRP-nonapeptide and BSA, the enzymatic reaction derived from labeled HRP on the H2O2-hydroquinone system make the electrochemical current of hydroquinone change, then the concentration of BSA can be indirectly determined. BSA in the range of 1.0–150 ng/mL exhibited a linear relationship with the differential pulse voltammetric current variation and the detection limit was 0.02 ng/mL. The sensor has high sensitivity, good selectivity, and reproducibility. It has been applied to the determination of residual bovine serum albumin in human rabies vaccine with the recovery rate of 98.3%–102.5%.

      PubDate: 2017-05-12T12:56:45Z
      DOI: 10.1016/j.ab.2017.05.006
      Issue No: Vol. 530 (2017)
       
  • Extending the throughput of Biacore 4000 biosensor to accelerate kinetic
           analysis of antibody-antigen interaction
    • Authors: Vishal Kamat; Ashique Rafique
      Pages: 75 - 86
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Vishal Kamat, Ashique Rafique
      The surface plasmon resonance (SPR) biosensors are being routinely used in different stages of drug discovery and development. However, the lack of high throughput SPR biosensors continues to be a primary bottleneck for the rapid kinetic screening of large panels of monoclonal antibodies (mAbs). To further increase the throughput of the Biacore 4000 biosensor, we have developed three kinetic screening assays to characterize mAb-antigen interactions – (i) 16-mAb capture kinetic, (ii) single cycle kinetic (SCK), and (iii) parallel kinetic (PK). The performance of all three kinetic assays was evaluated by characterizing the binding of kinetically diverse human mAbs to four antigens with molecular weights of 14kD, 29kD, 38kD, and 48kD and binding affinities ranging from 130pM to 200 nM. The binding rate constants measured using all three kinetic assays were reproducible across multiple experiments and correlated with the values generated using the conventional 8-mAb capture kinetic assay on the Biacore 4000 (R2 > 0.94). Moreover, the 16-mAb capture assay decreased experiment time and analyte consumption by 35% and 50%, respectively. This work illustrates the significance of the 16-mAb capture kinetic, SCK, and PK assays to increase the throughput of Biacore 4000 and to support rapid kinetic screening of mAbs.

      PubDate: 2017-05-12T12:56:45Z
      DOI: 10.1016/j.ab.2017.04.020
      Issue No: Vol. 530 (2017)
       
  • A sensitive chemiluminescence enzyme immunoassay based on molecularly
           imprinted polymers solid-phase extraction of parathion
    • Authors: Ge Chen; Maojun Jin; Pengfei Du; Chan Zhang; Xueyan Cui; Yudan Zhang; Yongxin She; Hua Shao; Fen Jin; Shanshan Wang; Lufei Zheng; Jing Wang
      Pages: 87 - 93
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Ge Chen, Maojun Jin, Pengfei Du, Chan Zhang, Xueyan Cui, Yudan Zhang, Yongxin She, Hua Shao, Fen Jin, Shanshan Wang, Lufei Zheng, Jing Wang
      The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74–18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C18. However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products.

      PubDate: 2017-05-17T13:11:25Z
      DOI: 10.1016/j.ab.2017.05.008
      Issue No: Vol. 530 (2017)
       
  • Long term kinetic measurements revealing precision and general performance
           of surface plasmon resonance biosensors
    • Authors: Franziska Steinicke; Imke Oltmann-Norden; Hermann Wätzig
      Pages: 94 - 103
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Franziska Steinicke, Imke Oltmann-Norden, Hermann Wätzig
      This work presents an extensive parameter list that facilitates a survey of biosensor performance using Biacore instruments for kinetic binding studies. Six long term measurements were performed using a strongly interacting antigen-antibody (β2 microglobulin) system. Both Single Cycle Kinetic (SCK) and Multi Cycle Kinetic (MCK) were executed each with five different analyte concentrations. The overall comparison of the long term monitored parameters, like the dissociation constant (KD with approximately 3–6% relative percental standard deviation), the association and dissociation rate constants (ka, kd), the analyte binding capacity (Rmax), chi2 and the sum of the absolute values of the residuals, revealed the delicate factors that make the system performance vulnerable. The main influential factors on kinetic performance were the regeneration conditions, the quality of the sensor surface, the usage time and alteration of the sensor surface, the dilution series and the number of run cycles (about 250–600 per chip). Moreover the direct comparison of MCK and SCK uncovered distinct differences in the accuracy of the KD values. The study of sensor chips from two manufacturers showed distinct differences in the precision of the data. Using control charts for the surveillance of these parameters contributes to an overall better system performance.

      PubDate: 2017-05-17T13:11:25Z
      DOI: 10.1016/j.ab.2017.05.009
      Issue No: Vol. 530 (2017)
       
  • An improved method to measure lipase activity in aqueous media
    • Authors: Samanta Hernández-García; María Inmaculada García-García; Francisco García-Carmona
      Pages: 104 - 106
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Samanta Hernández-García, María Inmaculada García-García, Francisco García-Carmona
      An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca+2 to be studied, while avoiding turbidity in the reaction medium.
      Graphical abstract image

      PubDate: 2017-05-17T13:11:25Z
      DOI: 10.1016/j.ab.2017.05.012
      Issue No: Vol. 530 (2017)
       
  • VEGFR1 domain 2 covalent labeling with horseradish peroxidase: Development
           of a displacement assay on VEGF
    • Authors: Marie Reille-Seroussi; Jean-François Gaucher; Laure-Anne Cussac; Isabelle Broutin; Michel Vidal; Sylvain Broussy
      Pages: 107 - 112
      Abstract: Publication date: 1 August 2017
      Source:Analytical Biochemistry, Volume 530
      Author(s): Marie Reille-Seroussi, Jean-François Gaucher, Laure-Anne Cussac, Isabelle Broutin, Michel Vidal, Sylvain Broussy
      The VEGFR1 has been shown to play a role in the regulation of angiogenesis, and has therefore been associated to several pathologies. In order to extend our toolbox of screening methods for the identification of compounds disrupting the VEGF receptor 1/VEGF interaction, we developed a fast and accurate displacement assay, in which VEGF receptor 1 domain 2 is directly labeled with an enzyme, bypassing the classical streptavidin-biotin interaction system. A description of this straightforward strategy is provided here, including its advantages and disadvantages. Optimization of the reagents preparation, purification and conservation, and displacement assay with known molecular entities are presented.

      PubDate: 2017-05-17T13:11:25Z
      DOI: 10.1016/j.ab.2017.05.004
      Issue No: Vol. 530 (2017)
       
  • Editorial for the special issue on introduction to in vivo Magnetic
           
    • Authors: Cristina Cudalbu; Arthur J.L. Cooper
      Pages: 1 - 3
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Cristina Cudalbu, Arthur J.L. Cooper


      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2017.05.014
      Issue No: Vol. 529 (2017)
       
  • B0 magnetic field homogeneity and shimming for in vivo magnetic
           resonance spectroscopy
    • Authors: Christoph Juchem; Robin A. de Graaf
      Pages: 17 - 29
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Christoph Juchem, Robin A. de Graaf
      The homogenization of B 0 conditions is necessary for every magnetic resonance spectroscopy (MRS) investigation. Its direct consequence is narrow spectral lines, on which reliable separation and quantification of biochemicals, and thus experimentally obtainable metabolic information, fundamentally relies. Besides spectral linewidth, unwanted B 0 inhomogeneity also impairs other aspects of the MRS experiment, such as water suppression and editing efficiency, that rely on exact frequency definition. Therefore, experimental B 0 homogenization, called B 0 shimming, is mandatory for meaningful MRS, and high-level B 0 shimming is arguably one of the most important ingredients for successful MRS investigations. In this review, we describe the relevance of B 0 homogeneity for in vivo MRS and summarize common concepts and specific solutions for its experimental optimization.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.06.003
      Issue No: Vol. 529 (2017)
       
  • A practical guide to in vivo proton magnetic resonance spectroscopy
           at high magnetic fields
    • Authors: Lijing Xin; Ivan Tkáč
      Pages: 30 - 39
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Lijing Xin, Ivan Tkáč
      Localized proton magnetic resonance spectroscopy (1H-MRS) is a noninvasive tool for measuring in vivo neurochemical information in animal and human brains. With the increase of magnetic field strength, whereas localized 1H-MRS benefits from higher sensitivity and spectral dispersion, it is challenged by increased spatial inhomogeneity of the B0 and B1 fields, larger chemical shift displacement error, and shortened T2 relaxation times of metabolites. Advanced localized 1H-MRS methodologies developed for high magnetic fields have shown promising results and allow the measurement of neurochemical profiles with up to 19 brain metabolites, including less-abundant metabolites, such as glutathione, glycine, γ-aminobutyric acid and ascorbate. To provide a practical guide for conducting in vivo 1H-MRS studies at high magnetic field strength, we reviewed various essential technical aspects from data acquisition (hardware requirements, B1 and B0 inhomogeneity, water suppression, localization sequences and acquisition strategies) to data processing (frequency and phase correction, spectral quality control, spectral fitting and concentration referencing). Additionally, we proposed guidelines for choosing the most appropriate data acquisition and processing approaches to maximize the achievable neurochemical information.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.10.019
      Issue No: Vol. 529 (2017)
       
  • Imaging based magnetic resonance spectroscopy (MRS) localization for
           quantitative neurochemical analysis and cerebral metabolism studies
    • Authors: Phil Lee; Peter Adany; In-Young Choi
      Pages: 40 - 47
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Phil Lee, Peter Adany, In-Young Choi
      Accurate quantitative metabolic imaging of the brain presents significant challenges due to the complexity and heterogeneity of its structures and compositions with distinct compartmentations of brain tissue types (e.g., gray and white matter). The brain is compartmentalized into various regions based on their unique functions and locations. In vivo magnetic resonance spectroscopy (MRS) techniques allow non-invasive measurements of neurochemicals in either single voxel or multiple voxels, yet the spatial resolution and detection sensitivity of MRS are significantly lower compared with MRI. A fundamentally different approach, namely spectral localization by imaging (SLIM) provides a new framework that overcomes major limitations of conventional MRS techniques. Conventional MRS allows only rectangular voxel shapes that do not conform to the shapes of brain structures or lesions, while SLIM allows compartments with arbitrary shapes. However, the restrictive assumption proposed in the original concept of SLIM, i.e., compartmental homogeneity, led to spectral localization errors, which have limited its broad applications. This review focuses on the recent technical frontiers of image-based MRS localization techniques that overcome the limitations of SLIM through the development and implementation of various new strategies, including incorporation of magnetic field inhomogeneity corrections, the use of multiple receiver coils, and prospective optimization of data acquisition.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2017.01.007
      Issue No: Vol. 529 (2017)
       
  • 1D-spectral editing and 2D multispectral in vivo 1H-MRS and 1H-MRSI -
           Methods and applications
    • Authors: Wolfgang Bogner; Gilbert Hangel; Morteza Esmaeili; Ovidiu C. Andronesi
      Pages: 48 - 64
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Wolfgang Bogner, Gilbert Hangel, Morteza Esmaeili, Ovidiu C. Andronesi
      This article reviews the methodological aspects of detecting low-abundant J-coupled metabolites via 1D spectral editing techniques and 2D nuclear magnetic resonance (NMR) methods applied in vivo, in humans, with a focus on the brain. A brief explanation of the basics of J-evolution will be followed by an introduction to 1D spectral editing techniques (e.g., J-difference editing, multiple quantum coherence filtering) and 2D-NMR methods (e.g., correlation spectroscopy, J-resolved spectroscopy). Established and recently developed methods will be discussed and the most commonly edited J-coupled metabolites (e.g., neurotransmitters, antioxidants, onco-markers, and markers for metabolic processes) will be briefly summarized along with their most important applications in neuroscience and clinical diagnosis.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.12.020
      Issue No: Vol. 529 (2017)
       
  • Optimization of metabolite detection by quantum mechanics simulations in
           magnetic resonance spectroscopy
    • Authors: Giulio Gambarota
      Pages: 65 - 78
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Giulio Gambarota
      Magnetic resonance spectroscopy (MRS) is a well established modality for investigating tissue metabolism in vivo. In recent years, many efforts by the scientific community have been directed towards the improvement of metabolite detection and quantitation. Quantum mechanics simulations allow for investigations of the MR signal behaviour of metabolites; thus, they provide an essential tool in the optimization of metabolite detection. In this review, we will examine quantum mechanics simulations based on the density matrix formalism. The density matrix was introduced by von Neumann in 1927 to take into account statistical effects within the theory of quantum mechanics. We will discuss the main steps of the density matrix simulation of an arbitrary spin system and show some examples for the strongly coupled two spin system.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.08.019
      Issue No: Vol. 529 (2017)
       
  • Quantum-mechanical simulations for in vivo MR spectroscopy: Principles
           and possibilities demonstrated with the program NMRScopeB
    • Authors: Zenon Starčuk; Jana Starčuková
      Pages: 79 - 97
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Zenon Starčuk, Jana Starčuková
      Current possibilities and limitations of the simulation of in vivo magnetic resonance spectroscopic signals are demonstrated from the point of view of a simulation software user as well as its programmer. A brief review of the quantum-mechanical background addresses the specific needs of simulation implementation and in vivo MR spectroscopy in general. Practical application examples demonstrate how flexible simulation software, such as NMRScopeB, can be utilized not only for the preparation of metabolite basis signals for quantification of metabolite concentrations, but also in pulse sequence development, assessment of artifacts and analyzing mechanism leading to unexpected signal phenomena.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.10.007
      Issue No: Vol. 529 (2017)
       
  • Quality management in in vivo proton MRS
    • Authors: Nuno Pedrosa de Barros; Johannes Slotboom
      Pages: 98 - 116
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Nuno Pedrosa de Barros, Johannes Slotboom
      The quality of MR-Spectroscopy data can easily be affected in in vivo applications. Several factors may produce signal artefacts, and often these are not easily detected, not even by experienced spectroscopists. Reliable and reproducible in vivo MRS-data requires the definition of quality requirements and goals, implementation of measures to guarantee quality standards, regular control of data quality, and a continuous search for quality improvement. The first part of this review includes a general introduction to different aspects of quality management in MRS. It is followed by the description of a series of tests and phantoms that can be used to assure the quality of the MR system. In the third part, several methods and strategies used for quality control of the spectroscopy data are presented. This review concludes with a reference to a few interesting techniques and aspects that may help to further improve the quality of in vivo MR-spectra.

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2017.01.017
      Issue No: Vol. 529 (2017)
       
  • Technical and experimental features of Magnetic Resonance Spectroscopy of
           brain glycogen metabolism
    • Authors: Ana Francisca Soares; Rolf Gruetter; Hongxia Lei
      Pages: 117 - 126
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Ana Francisca Soares, Rolf Gruetter, Hongxia Lei
      In the brain, glycogen is a source of glucose not only in emergency situations but also during normal brain activity. Altered brain glycogen metabolism is associated with energetic dysregulation in pathological conditions, such as diabetes or epilepsy. Both in humans and animals, brain glycogen levels have been assessed non-invasively by Carbon-13 Magnetic Resonance Spectroscopy (13C-MRS) in vivo. With this approach, glycogen synthesis and degradation may be followed in real time, thereby providing valuable insights into brain glycogen dynamics. However, compared to the liver and muscle, where glycogen is abundant, the sensitivity for detection of brain glycogen by 13C-MRS is inherently low. In this review we focus on strategies used to optimize the sensitivity for 13C-MRS detection of glycogen. Namely, we explore several technical perspectives, such as magnetic field strength, field homogeneity, coil design, decoupling, and localization methods. Furthermore, we also address basic principles underlying the use of 13C-labeled precursors to enhance the detectable glycogen signal, emphasizing specific experimental aspects relevant for obtaining kinetic information on brain glycogen.
      Graphical abstract image

      PubDate: 2017-06-12T14:20:41Z
      DOI: 10.1016/j.ab.2016.12.023
      Issue No: Vol. 529 (2017)
       
  • Introduction to nuclear magnetic resonance
    • Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Vladimír Mlynárik
      Nuclear magnetic resonance spectroscopy is a useful tool for studying normal and pathological biochemical processes in tissues. In this review, the principles of nuclear magnetic resonance and methods of obtaining nuclear magnetic resonance spectra are briefly outlined. The origin of the most important spectroscopic parameters—chemical shifts, coupling constants, longitudinal and transverse relaxation times, and spectroscopic line intensities—is explained, and the role of these parameters in interpretation of spectra is addressed. Basic methodological concepts of localized spectroscopy and spectroscopic imaging for the study of tissue metabolism in vivo are also described.

      PubDate: 2017-06-12T14:20:41Z
       
  • Radio-frequency coils for ultra-high field magnetic resonance
    • Authors: Ipek
      Abstract: Publication date: 15 July 2017
      Source:Analytical Biochemistry, Volume 529
      Author(s): Özlem Ipek
      Radiofrequency (RF) coils are key components of magnetic resonance imaging (MRI) systems. The primary purpose of this review is to provide a basic theory of RF coil designs and their characterization by bench measurements, electromagnetic field simulations and MR measurements. With the continuing increase of magnetic field strength in MRI instruments, the RF wavelength in the subject under study becomes comparable to or smaller in size than the anatomical dimensions of the tissue under study, which amplifies the signal inhomogeneity. Also, RF energy increases quadratically with the Larmor frequency, which leads to increased heat deposition in the subject, especially at ultra-high field. Elegant RF coil designs are explored here to address these challenges.

      PubDate: 2017-06-12T14:20:41Z
       
  • Dansylglycine, a fluorescent probe for specific determination of
           halogenating activity of myeloperoxidase and eosinophil peroxidase
    • Authors: Luiza de Carvalho Bertozo; Maria Luiza Zeraik; Valdecir Farias Ximenes
      Abstract: Publication date: Available online 3 June 2017
      Source:Analytical Biochemistry
      Author(s): Luiza de Carvalho Bertozo, Maria Luiza Zeraik, Valdecir Farias Ximenes
      Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl−) and bromide (Br−), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H2O2) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H2O2. The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3′,5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr.

      PubDate: 2017-06-06T12:06:07Z
      DOI: 10.1016/j.ab.2017.05.029
       
 
 
JournalTOCs
School of Mathematical and Computer Sciences
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