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Showing 1 - 200 of 3043 Journals sorted alphabetically
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 20, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 18, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 83, 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: 331, 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   (Followers: 1)
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: 211, 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: 23, SJR: 1.365, h-index: 73)
Acta Sociológica     Open Access  
Acta Tropica     Hybrid Journal   (Followers: 6, 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: 8, SJR: 1.039, h-index: 5)
Additives for Polymers     Full-text available via subscription   (Followers: 20)
Advanced Drug Delivery Reviews     Hybrid Journal   (Followers: 128, 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: 22, 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: 10, 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: 41, 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: 47, SJR: 0.674, h-index: 38)
Advances in Fuel Cells     Full-text available via subscription   (Followers: 15)
Advances in Genetics     Full-text available via subscription   (Followers: 15, SJR: 2.558, h-index: 54)
Advances in Genome Biology     Full-text available via subscription   (Followers: 11)
Advances in Geophysics     Full-text available via subscription   (Followers: 6, SJR: 2.325, h-index: 20)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 21, 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: 25)
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: 35, 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: 5)
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: 6, 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: 24, 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: 60)
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: 343, 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: 7, SJR: 0.823, h-index: 27)
Advances in the Study of Behavior     Full-text available via subscription   (Followers: 30, 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: 307, 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: 8, SJR: 3.289, h-index: 78)
Aggression and Violent Behavior     Hybrid Journal   (Followers: 405, 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: 53, 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: 9, 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: 4, 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: 7, SJR: 0.158, h-index: 9)
Alzheimer's & Dementia     Hybrid Journal   (Followers: 48, 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: 48, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 45, SJR: 2.063, h-index: 186)
American J. of Emergency Medicine     Hybrid Journal   (Followers: 38, 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: 16, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 31, 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: 33, SJR: 2.313, h-index: 172)
American J. of Medicine     Hybrid Journal   (Followers: 46, 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: 191, 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: 3)
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: 26, 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: 34, 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: 55, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 10)
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: 162, 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: 11)
Anesthésie & Réanimation     Full-text available via subscription   (Followers: 1)
Anesthesiology Clinics     Full-text available via subscription   (Followers: 22, 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: 157, 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   (Followers: 1, 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]   [162 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3043 journals]
  • Isolation of mouse chromaffin secretory vesicles and their division into
           12 fractions
    • Authors: Marta R. Pardo; Judith Estévez-Herrera; Leandro Castañeyra; Ricardo Borges; José David Machado
      Pages: 1 - 7
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Marta R. Pardo, Judith Estévez-Herrera, Leandro Castañeyra, Ricardo Borges, José David Machado
      The study of chromaffin secretory vesicles (SVs) has contributed immensely to our understanding of exocytosis. These organelles, also called chromaffin granules, are a specific type of large dense secretory vesicle found in many endocrine cells and neurons. Traditionally, they have been isolated from bovine adrenal glands due to the large number of SVs that can be obtained from this tissue. However, technical advances now make it possible to obtain very pure preparations of SVs from mice, which is particular interesting for functional studies given the availability of different genetically modified strains of mice. Despite the small size of the mouse adrenal medulla (400–500 μm and less than 2 mg in weight), we have successfully carried out functional studies on SVs isolated from WT and knockout mice. As such, we present here our method to purify crude vesicles and to fractionate mouse chromaffin SVs, along with examples of their functional characterization.

      PubDate: 2017-08-15T19:30:59Z
      DOI: 10.1016/j.ab.2017.07.026
      Issue No: Vol. 536 (2017)
  • Biosensors based on β-galactosidase enzyme: Recent advances and
    • Authors: Shiv K. Sharma; Roger M. Leblanc
      Pages: 1 - 11
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Shiv K. Sharma, Roger M. Leblanc
      Many industries are striving for the development of more reliable and robust β-galactosidase biosensors that exhibit high response rate, increased detection limit and enriched useful lifetime. In a newfangled technological atmosphere, a trivial advantage or disadvantage of the developed biosensor may escort to the survival and extinction of the industry. Several alternative strategies to immobilize β-galactosidase enzyme for their utilization in biosensors have been developed in recent years in the quest of maximum utility by controlling the defects seen in the previous biosensors. The overwhelming call for on-line measurement of different sample constituents has directed science and industry to search for best practical solutions and biosensors are witnessed as the best prospect. The main objective of this paper is to serve as a narrow footbridge by comparing the literary works on the β-galactosidase biosensors, critically analyze their use in the construction of best biosensor by showing the pros and cons of the predicted methods for the practical use of biosensors.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.019
      Issue No: Vol. 535 (2017)
  • An enzyme-linked immunosorbent assay for the detection of diacetyl
    • Authors: Lucia Marri; Anita M. Jansson; Caspar E. Christensen; Ole Hindsgaul
      Pages: 12 - 18
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Lucia Marri, Anita M. Jansson, Caspar E. Christensen, Ole Hindsgaul
      Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.021
      Issue No: Vol. 535 (2017)
  • Colorimetric sensing of selenocystine using gold nanoparticles
    • Authors: Liyang Liu; Xia Wang; Juan Yang; Yan Bai
      Pages: 19 - 24
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Liyang Liu, Xia Wang, Juan Yang, Yan Bai
      We present a highly selective and sensitive colorimetric method for the detection of selenocystine (SeCys) coexisting with other amino acids, especially cysteine (Cys) using the gold nanoparticles (AuNPs). Firstly, Cys was oxidized to cystine (Cys-Cys) by dissolved oxygen under Cu2+ catalysis in the pre-reaction, which eliminated the interference of Cys in the SeCys sensing process. Then SeCys induced the rapid aggregation of AuNPs through Au-Se bond and complex formation of Cu2+-SeCys in the colorimetric reaction, in which the color change of AuNPs from red to blue or purple with the naked eye detection or with a UV-vis spectrophotometric determination. The concentration of SeCys was quantified by the value at 670 nm from the second-derivative SPR absorbance spectrum. The linear range was from 2 μM to 14 μM with correlation coefficient of 0.999 and a detection limit (LOD) was 0.14 μM. Moreover, the colorimetric response of AuNPs exhibited remarkable specificity to SeCys coexisting with 18 amino acids in a simulation sample, in which the total concentration of Cys and Cys-Cys was less than 15 μM and the each concentration of other 16 common amino acids was 10 μM.
      Graphical abstract image

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.020
      Issue No: Vol. 535 (2017)
  • Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in
           three-dimensional cell culture
    • Authors: Peter Worthington; Katherine M. Drake; Zhiqin Li; Andrew D. Napper; Darrin J. Pochan; Sigrid A. Langhans
      Pages: 25 - 34
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Peter Worthington, Katherine M. Drake, Zhiqin Li, Andrew D. Napper, Darrin J. Pochan, Sigrid A. Langhans
      Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that β-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 β-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.024
      Issue No: Vol. 535 (2017)
  • ESCORTing proteins directly from whole cell-lysate for single-molecule
    • Authors: Shwetha Srinivasan; Jagadish P. Hazra; Gayathri S. Singaraju; Debadutta Deb; Sabyasachi Rakshit
      Pages: 35 - 42
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Shwetha Srinivasan, Jagadish P. Hazra, Gayathri S. Singaraju, Debadutta Deb, Sabyasachi Rakshit
      We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.022
      Issue No: Vol. 535 (2017)
  • Neutral Red versus MTT assay of cell viability in the presence of copper
    • Authors: Mariela Gomez Perez; Lyvia Fourcade; Mircea Alexandru Mateescu; Joanne Paquin
      Pages: 43 - 46
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Mariela Gomez Perez, Lyvia Fourcade, Mircea Alexandru Mateescu, Joanne Paquin
      Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu(II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.027
      Issue No: Vol. 535 (2017)
  • Advances in methods for characterization of hepatic urea cycle enzymatic
           activity in HepaRG cells using UPLC-MS/MS
    • Authors: M.F. Moedas; A.A.A. Adam; M.A. Farelo; L. IJlst; R.A.F.M. Chamuleau; R. Hoekstra; R.J.A. Wanders; M.F.B. Silva
      Pages: 47 - 55
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): M.F. Moedas, A.A.A. Adam, M.A. Farelo, L. IJlst, R.A.F.M. Chamuleau, R. Hoekstra, R.J.A. Wanders, M.F.B. Silva
      Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.
      Graphical abstract image

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.025
      Issue No: Vol. 535 (2017)
  • Electrochemical detection of interaction between capsaicin and nucleic
           acids in comparison to agarose gel electrophoresis
    • Authors: Nilay Yilmaz; Ece Eksin; Bilge Karacicek; Yasemin Eraç; Arzum Erdem
      Pages: 56 - 62
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Nilay Yilmaz, Ece Eksin, Bilge Karacicek, Yasemin Eraç, Arzum Erdem
      In this study, the biomolecular interaction occurred between nucleic acids and Capsaicin (CPS), the active compound in chilli peppers, which has been reported to have anti-carcinogenic properties, was investigated for the first time herein using disposable electrochemical biosensor. It is aimed to perform the surface-confined interaction between CPS and different types of nucleic acids and under this aim, the experimental conditions were optimized; such as, the concentration of CPS and DNA, DNA immobilization time and interaction time etc. The detection limit of DNA was estimated based on guanine oxidation signal in the linear concentration range of DNA from 1 to 5 μg/mL, and it was found to be 0.62 μg/mL. The effect of time-dependent manner from 1 min to 30 min on the interaction of CPS with nucleic acids was explored upon to the changes at guanine signal coming from double stranded DNA and cDNA as well as PCR samples. The interaction of CPS with double stranded DNA was also determined by agarose gel electrophoresis.
      Graphical abstract image

      PubDate: 2017-08-15T19:30:59Z
      DOI: 10.1016/j.ab.2017.07.023
      Issue No: Vol. 535 (2017)
  • A colorimetric sensor of cysteine based on self-assembly nanostructures of
           Fe3+-H2O2/Tetramethylbenzidine system with “On-Off” switching function
    • Authors: Zhonghua Xue; Xiaofen Wang; Honghong Rao; Xiuhui Liu; Xiaoquan Lu
      Pages: 1 - 9
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Zhonghua Xue, Xiaofen Wang, Honghong Rao, Xiuhui Liu, Xiaoquan Lu
      Many strategies have been explored for selectively and sensitively detecting cysteine in different samples. Here, a novel colorimetric sensor based on self-assembly nanostructures of Fe3+-H2O2/Tetramethylbenzidine system with dual-level logic gate function and colorimetric determination of cysteine were firstly explored. The proposed Fe3+-H2O2-TMB system provides a sensitive optical signal due to the selectively reductive ability of cysteine to the oxidized TMB and thus could be successfully applied to the construction of instant on-site visual detection method with a paper based test strip for cysteine determination in a sample solution as well as for a dual-level logic gate fabrication.
      Graphical abstract image

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.003
      Issue No: Vol. 534 (2017)
  • Biolayer interferometry predicts ELISA performance of monoclonal antibody
           pairs for Plasmodium falciparum histidine-rich protein 2
    • Authors: C.F. Markwalter; I.K. Jang; R.A. Burton; G.J. Domingo; D.W. Wright
      Pages: 10 - 13
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): C.F. Markwalter, I.K. Jang, R.A. Burton, G.J. Domingo, D.W. Wright
      Predicting antibody pair performance in a sandwich format streamlines development of antibody-based diagnostics and laboratory research tools, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFAs). We have evaluated panels of monoclonal antibodies against the malarial parasite biomarker Plasmodium falciparum histidine rich protein 2 (HRP2), including 9 new monoclonal antibodies, using biolayer interferometry (BLI) and screened antibody pairs in a checkerboard ELISA. This study showed BLI predicts antibody pair ELISA performance for HRP2. Pairs that included capture antibodies with low off-rate constants and detection antibodies with high on-rate constants performed best in an ELISA format.
      Graphical abstract image

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.010
      Issue No: Vol. 534 (2017)
  • Polydopamine-immobilized polypropylene microfuge tube as a pH-responsive
           platform for capture/release of DNA from foodborne pathogens
    • Authors: Zitao Zhong; Xin Yao; Xiaomei Gao; Li Jia
      Pages: 14 - 18
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Zitao Zhong, Xin Yao, Xiaomei Gao, Li Jia
      A rapid, convenient and efficient DNA extraction method with no need for toxic agents and centrifugation was reported. A polypropylene microfuge (MF) tube was used as the substrate to immobilize polydopamine (PDA). The prepared PDA-coated MF (PDA@MF) tube was used as a pH-responsive platform for rapid extraction of DNA based on pH-induced charge switch of amino and phenolic hydroxyl groups in PDA coating. The extraction procedure is simple and can be finished in 25 min. The PDA@MF tube was applied for extraction of genomic DNA from foodborne pathogens in milk. The extracted DNA was directly used as template for PCR amplification.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.004
      Issue No: Vol. 534 (2017)
  • Quantitation of CRM197 using imaged capillary isoelectric focusing with
           fluorescence detection and capillary Western
    • Authors: John W. Loughney; Sha Ha; Richard R. Rustandi
      Pages: 19 - 23
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): John W. Loughney, Sha Ha, Richard R. Rustandi
      Maurice is a new instrument that can perform imaged capillary isoelectric focusing (icIEF). The standard detection for icIEF is UV absorbance at 280 nm, which limits its application to high protein concentration samples and non-complex samples. Here we describe an icIEF instrument with fluorescence detection. We demonstrate the advantage of using either icIEF with fluorescence detection or quantitative Western Blot to measure diphtheria toxin mutant CRM197 protein titer in crude cell lysates and purified samples. These two techniques have great potentials to become standard methods to analyze protein titers in crude cell lysate or other complex samples types.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.06.013
      Issue No: Vol. 534 (2017)
  • Hydrogen peroxide agarose gels for electrophoretic analysis of RNA
    • Authors: Renu Pandey; Daman Saluja
      Pages: 24 - 27
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Renu Pandey, Daman Saluja
      Efficient electrophoretic separation of isolated total RNA utilizes chemicals and agents to aid in nuclease free environment. However cost, extensive pre-run processing protocols as well as toxic byproducts limit the usage of such protocols. Moreover, these treatments affect the overall electrophoretic results by altering the conductivity of the running buffer and weaken the gel strength. We here provide a protocol for RNA visualization that obviates these shortcomings by preparation of agarose gel with hydrogen peroxide using the regular TAE buffer. The simple, inexpensive protocol exhibits superior results in a horizontal agarose gel electrophoresis.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.007
      Issue No: Vol. 534 (2017)
  • A rapid mass spectrometric method for the measurement of catalytic
           activity of ten-eleven translocation enzymes
    • Authors: Babu Sudhamalla; Debasis Dey; Megan Breski; Kabirul Islam
      Pages: 28 - 35
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Babu Sudhamalla, Debasis Dey, Megan Breski, Kabirul Islam
      Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (K M and k cat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by β-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.
      Graphical abstract image

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.06.011
      Issue No: Vol. 534 (2017)
  • Red-emitting chimeric firefly luciferase for in vivo imaging in low
           ATP cellular environments
    • Authors: Bruce R. Branchini; Tara L. Southworth; Danielle M. Fontaine; Dawn Kohrt; Franceine S. Welcome; Catherine M. Florentine; Emma R. Henricks; Demetria B. DeBartolo; Elisa Michelini; Luca Cevenini; Aldo Roda; Martha J. Grossel
      Pages: 36 - 39
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Bruce R. Branchini, Tara L. Southworth, Danielle M. Fontaine, Dawn Kohrt, Franceine S. Welcome, Catherine M. Florentine, Emma R. Henricks, Demetria B. DeBartolo, Elisa Michelini, Luca Cevenini, Aldo Roda, Martha J. Grossel
      Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high K m values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a K m value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the K m value in low ATP environments.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.001
      Issue No: Vol. 534 (2017)
  • Prediction of protein N-formylation using the composition of k-spaced
           amino acid pairs
    • Authors: Zhe Ju; Jun-Zhe Cao
      Pages: 40 - 45
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Zhe Ju, Jun-Zhe Cao
      As one of important protein post-translational modifications, N-formylation has been reported to be involved in various biological processes. The accurate identification of N-formylation sites is crucial for understanding the underlying mechanisms of N-formylation. Since the traditional experimental methods are generally labor-intensive and expensive, it is important to develop computational methods to predict N-formylation sites. In this paper, a predictor named NformPred is proposed to improve the prediction of N-formylation sites by using composition of k-spaced amino acid pairs encoding scheme and support vector machine algorithm. As illustrated by 10-fold cross-validation, NformPred achieves a promising performance with a Sensitivity of 86.00%, a Specificity of 96.25%, an Accuracy of 94.48% and a Matthew's correlation coefficient of 0.8099, which are much better than those of current computational method. Feature analysis shows that some k-spaced amino acid pairs such as ‘IxxL’, ‘LV’ and ‘IxxxI’ play the most important roles in the prediction of N-formylation sites. These predictive and analytical results suggest that NformPred might facilitate the identification of protein N-formylation. A free online service for NformPred is accessible at

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.011
      Issue No: Vol. 534 (2017)
  • Rapid preparation of adherent mammalian cells for basic scanning electron
           microscopy (SEM) analysis
    • Authors: Andrew Osahor; Karthik Deekonda; Lee Choon Weng; Edmund Ui-Hang Sim; Aurelian Radu; Kumaran Narayanan
      Pages: 46 - 48
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Andrew Osahor, Karthik Deekonda, Lee Choon Weng, Edmund Ui-Hang Sim, Aurelian Radu, Kumaran Narayanan
      Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.008
      Issue No: Vol. 534 (2017)
  • High-throughput quantitation of Fc-containing recombinant proteins in cell
           culture supernatant by fluorescence polarization spectroscopy
    • Authors: Ben Thompson; Jerry Clifford; Mike Jenns; Andrew Smith; Ray Field; Kalpana Nayyar; David C. James
      Pages: 49 - 55
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Ben Thompson, Jerry Clifford, Mike Jenns, Andrew Smith, Ray Field, Kalpana Nayyar, David C. James
      Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (<2 min per 96 samples) 96-well microplate-based assay that enables high-throughput quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5–80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of <1%, and a between-plate standard deviation below 1%. Comparative assays of the same samples by FP and other commonly used IgG assay formats operating over a similar dynamic range (Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.013
      Issue No: Vol. 534 (2017)
  • A Fe3O4@Au-basedpseudo-homogeneous electrochemical immunosensor for AFP
           measurement using AFP antibody-GNPs-HRP as detection probe
    • Authors: Yulin Yuan; Shanshan Li; Yewei Xue; Jintao Liang; Lijie Cui; Qingbo Li; Sufang Zhou; Yong Huang; Guiyin Li; Yongxiang Zhao
      Pages: 56 - 63
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Yulin Yuan, Shanshan Li, Yewei Xue, Jintao Liang, Lijie Cui, Qingbo Li, Sufang Zhou, Yong Huang, Guiyin Li, Yongxiang Zhao
      In this study, a Fe3O4@Au-based pseudo-homogeneous electrochemical immunosensor was prepared for detection of alpha fetoprotein (AFP), a well-known hepatocellular carcinoma biomarker. The primary antibody (Ab1) was immobilized on Fe3O4@Au NPs as the capture probe. Horseradish peroxidase (HRP) and secondary antibody (Ab2) were conjugated on gold nanoparticles (GNPs) through electrostatic adsorption to form signal-amplifying labels. In the presence of AFP, a sandwich immunocomplex was formed via specific recognition of antigen-antibody in a Fe3O4@Au-basedpseudo-homogeneousreaction system. After the immunocomplex was captured to the surface of magnetic glassy carbon electrode (MGCE), the labeling HRP catalyzed the decomposition of H2O2, resulting in a substantial current for the quantitative detection of AFP. The amperometric (i-t) method was employed to record the response signal of the immunosensor based on the catalysis of the immobilized HRP toward the reduction of H2O2 with hydroquinone (HQ) as the redox mediator. Under the optimal conditions, the amperometric current response presented a linear relationship with AFP concentration over the range of 20 ng/mL-100 ng/mLwith a correlation coefficient of 0.9940, and the detection limit was 0.64 ng/mL at signal/noise [S/N] = 3. Moreover, the electrochemical immunosensor exhibited higher anti-interference ability, acceptable reproducibility and long-term stability for AFP detection.
      Graphical abstract image

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.015
      Issue No: Vol. 534 (2017)
  • Ultra-sensitive aptasensor based on a GQD nanocomposite for detection of
           hepatitis C virus core antigen
    • Authors: Kazhal Ghanbari; Mahmoud Roushani; Azadeh Azadbakht
      Pages: 64 - 69
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Kazhal Ghanbari, Mahmoud Roushani, Azadeh Azadbakht
      In the present study, by using the aptamer proximity binding assay strategy, a novel electrochemical aptasensor is described for ultrasensitive detection of hepatitis C virus (HCV) core antigen. The immobilization surface is prepared by the modification of a glassy carbon electrode (GCE) with a graphene quantum dots (GQD). GQD were introduced as a novel and suitable substrate for aptamers through π-π stacking interactions, the richness of hydrophilic edges as well as hydrophobic plane in GQD which enhances the aptamer absorption on the electrode surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed at each stage of the chemical modification process to confirm the resulting surface changes. EIS technique was used as an efficient alternative detection system for HCV core antigen measurement with detection limit 3.3 pg mL−1 and two linear concentration range 10–70 pg mL−1 and 70–400 pg mL−1. Moreover, the fabricated aptasensor could accurately detect HCV core antigen concentration in human serum samples. Such an aptasensor opens a rapid, selective and sensitive route for HCV core antigen detection and provides a promising strategy for potential applications in clinical diagnostics.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.016
      Issue No: Vol. 534 (2017)
  • Loop-mediated isothermal amplification in disposable polyester-toner
    • Authors: Kezia Gomes de Oliveira; Juliane Cristina Borba; Alexandre Melo Bailão; Célia Maria de Almeida Soares; Emanuel Carrilho; Gabriela Rodrigues Mendes Duarte
      Pages: 70 - 77
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Kezia Gomes de Oliveira, Juliane Cristina Borba, Alexandre Melo Bailão, Célia Maria de Almeida Soares, Emanuel Carrilho, Gabriela Rodrigues Mendes Duarte
      In recent years, isothermal DNA amplification methods have emerged as an alternative in molecular diagnostics due to its ease of operation. The purpose of using isothermal amplification is to simplify the diagnostics work by i) eliminating heating cycles, ii) reducing equipment costs, and iii) providing rapid and accurate results in laboratories with limited resources. Here we show a simple and fast method for E. coli detection in disposable polyester-toner (PeT) microdevice. The amplification by LAMP of the malB gene from E. coli was carried out in a microchamber with 5-μL capacity and the reaction was thermally controlled with a thermoblock at 66 °C for 60 min. The passivation of the surface of PeT channels with BSA improved the efficiency of the LAMP reaction. The detection of amplified LAMP fragments was performed directly on-chip by visual detection and validated with off-chip detection to compare results. Visualization of amplicons directly in the microchip yielded positive reactions as low as 10 double-stranded DNA copies. Separation by gel electrophoresis was able to detect amplicons in reactions that initiated only with one copy of double-stranded DNA. We demonstrate that LAMP in PeT microchip is an important tool for molecular diagnostics at the point-of-care.

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.014
      Issue No: Vol. 534 (2017)
  • Using Au@nano-C60 nanocomposite as an enhanced sensing platform in
           modeling a TNT aptasensor
    • Authors: Mahmoud Roushani; Faezeh Shahdost-fard; Azadeh Azadbakht
      Pages: 78 - 85
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Mahmoud Roushani, Faezeh Shahdost-fard, Azadeh Azadbakht
      Based on the unique characteristics of the combination of fullerene and gold nanoparticles, we successfully designed a new and facile nanocomposite (Au@nano-C60) to fabricate an aptasensor for the ultra-sensitive and selective detection of TNT. The gold nanoparticles decorated fullerene onto a glassy carbon electrode was prepared using an electrochemical method by the in situ generation of Au nanoparticles onto the surface of the glassy carbon electrode modified with activated fullerene. Successively, the NH2-Apt as a receptor molecule of 2,4,6-Trinitrotoluen was covalently attached onto the modified electrode surface with the resultant nanocomposite. With the addition of the target onto the aptasensor surface and the formation of target/Apt complex, a linear response was obtained from 0.50 fM to 5 μM as well as a limit of detection down to 0.17 fM. The proposed aptasensor shows a wider linear response range and lower limit of detection for the specific detection of 2,4,6-Trinitrotoluen. This newly developed strategy will pave the way to partly meet the requirements in the field of homeland security and public safety.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.017
      Issue No: Vol. 534 (2017)
  • Obtaining a high activity subtilisin preparation by controlled thermal
           stress in n-octane
    • Authors: Shivcharan Prasad; Ipsita Roy
      Pages: 86 - 90
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Shivcharan Prasad, Ipsita Roy
      The use of enzymes in organic solvents has considerably widened their repertoire of applications. Such low water containing media also offer the possibility of carrying out enzymatic reactions at higher temperatures and enhancing reaction yields. The utility of such preparations is limited by the damage caused to the protein structure during freeze-drying. This work investigates the result of exposing the proteolytic enzyme subtilisin to high temperature in low water containing n-octane on its activity in aqueous and non-aqueous media. Exposing subtilisin at 90 °C for 5 h led to 18-fold improvement in its transesterification activity even at the normal assay temperature (37 °C) when compared with the untreated enzyme. The use of n-octane as the reaction medium was important as it helped to retain the three-dimensional architecture of the enzyme and should be considered while designing strategies for obtaining high activity preparations of other enzymes. Structural analysis using differential scanning fluorimetry showed that the enzyme lost its structure after heating in aqueous medium but retained it when heated in organic solvent. The simplicity and general applicability of the strategy should make it useful for obtaining highly active preparations of other enzymes as well.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.018
      Issue No: Vol. 534 (2017)
  • High-throughput estimation of specific activities of enzyme/mutants in
           cell lysates through immunoturbidimetric assay of proteins
    • Authors: Xiaolan Yang; Yiran Feng; Huimin Chong; Deqiang Wang; Xiaolei Hu; Jun Pu; Chang-Guo Zhan; Fei Liao
      Pages: 91 - 98
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Xiaolan Yang, Yiran Feng, Huimin Chong, Deqiang Wang, Xiaolei Hu, Jun Pu, Chang-Guo Zhan, Fei Liao
      High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 μg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.05.015
      Issue No: Vol. 534 (2017)
  • Development of carbon−graphene-based aptamer biosensor for EN2
           protein detection
    • Authors: Kalpana Settu; Jen-Tsai Liu; Ching-Jung Chen; Jang-Zern Tsai
      Pages: 99 - 107
      Abstract: Publication date: 1 October 2017
      Source:Analytical Biochemistry, Volume 534
      Author(s): Kalpana Settu, Jen-Tsai Liu, Ching-Jung Chen, Jang-Zern Tsai
      In this study, we developed a screen-printed carbon−graphene-based electrochemical biosensor for EN2 protein detection. The engrailed-2 (EN2) protein, a biomarker for prostate cancer, is known to be a strong binder to a specific DNA sequence (5′-TAATTA-3′) to regulate transcription. To take advantage of this intrinsic property, aptamer probes with TAATTA sequence was immobilized onto the screen-printed carbon−graphene electrode surface via EDC−NHS coupling approach. Cyclic voltammetry (CV) of the electrochemical measurement technique was employed for the quantitative detection of EN2 protein. The hindrance to the redox reaction of potassium ferricyanide on the biosensor surface due to the binding of the immobilized aptamer with its target EN2 protein quantified the protein concentration. Under optimum conditions, the aptamer biosensor can detect EN2 protein over a linear range from 35 to 185 nM with a detection limit of 38.5 nM.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.012
      Issue No: Vol. 534 (2017)
  • A brief history of Analytical Biochemistry: An appreciation for Dr.
           William Jakoby, Editor-in-Chief from 1986 to 2017
    • Authors: Arthur J.L. Cooper
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Arthur J.L. Cooper

      PubDate: 2017-07-21T13:08:31Z
      DOI: 10.1016/j.ab.2017.07.002
      Issue No: Vol. 533 (2017)
  • 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)
  • Localized degradation of foreign DNA strands in cells: Only excising the
           first nucleotide of 5′ region
    • Authors: Hui Li; Wei Shen; Michael Hon-Wah Lam; Haojun Liang
      Pages: 10 - 17
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Hui Li, Wei Shen, Michael Hon-Wah Lam, Haojun Liang
      Intracellular delivery of foreign DNA probes sharply increases the efficiency of various biodetection protocols. Spherical nucleic acid (SNA) conjugate is a new type of probe that consists of a dense oligonucleotide shell attached typically to a gold nanoparticle core. They are widely used as novel labels for in vitro biodetection and intracellular assay. However, the degradation of foreign DNA still remains a challenge that can cause significant signal leakage (false positive signal). Hence, the site and behavior of intracellular degradation need to be investigated. Herein, we discover a localized degradation behavior that only excises the first nucleotide of 5′ terminal from a DNA strand, whereas the residual portion of this strand is unbroken in MCF-7 cell. This novel degradation action totally differs from previous opinion that foreign DNA strand would be digested into tiny fragments or even individual nucleotides in cellular environment. On the basis of these findings, we propose a simple and effective way to avoid degradation-caused false positive that one can bypass the degradable site and choose a secure region to label fluorophore along the DNA stand, when using DNA probes for intracellular biodetection.

      PubDate: 2017-07-01T14:29:50Z
      DOI: 10.1016/j.ab.2017.04.006
      Issue No: Vol. 533 (2017)
  • Aptasensors for quantitative detection of Salmonella Typhimurium
    • Authors: Najmeh Ansari; Rezvan Yazdian-Robati; Mahin Shahdordizadeh; Zhouping Wang; Kiarash Ghazvini
      Pages: 18 - 25
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Najmeh Ansari, Rezvan Yazdian-Robati, Mahin Shahdordizadeh, Zhouping Wang, Kiarash Ghazvini
      Salmonella is one of the most frequent causes of food borne infectious disease. Among nearly 2500 documented serotypes are reported, Salmonella Typhimurium is the number one serotype associated with salmonellosis worldwide. Many different methods have been developed for the detection and quantification of S. typhimurium. Most of these assays are usually expensive, time consuming and require difficult sample preparation steps. Therefore, it is necessary to develop rapid, robust, cost-effective and sensitive alternative detection methods. In the last years, aptasensors, used for detection of S. typhimurium in different samples. In this review, recent advances and applications of aptasensors for the detection and quantification of S. typhimurium in details have been summarized.

      PubDate: 2017-07-01T14:29:50Z
      DOI: 10.1016/j.ab.2017.06.008
      Issue No: Vol. 533 (2017)
  • On-nylon membrane detection of nucleic acid molecules by rolling circle
    • Authors: Xinhui Xu; Beibei Zhang; Ping Gan; Jian Wu; Wei Dai; Ling Zhang; Jinke Wang
      Pages: 26 - 33
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Xinhui Xu, Beibei Zhang, Ping Gan, Jian Wu, Wei Dai, Ling Zhang, Jinke Wang
      Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3′ end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.

      PubDate: 2017-07-01T14:29:50Z
      DOI: 10.1016/j.ab.2017.06.005
      Issue No: Vol. 533 (2017)
  • An aptamer-based PCR method coupled with magnetic immunoseparation for
           sensitive detection of Salmonella Typhimurium in ground turkey
    • Authors: Lijun Wang; Ronghui Wang; Hong Wang; Michael Slavik; Hua Wei; Yanbin Li
      Pages: 34 - 40
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Lijun Wang, Ronghui Wang, Hong Wang, Michael Slavik, Hua Wei, Yanbin Li
      Aptamers are single-stranded oligonucleotide ligands that can bind to targets with high affinity and specificity. They have been widely studied in the field of diagnostics as alternatives to antibodies due to their favorable features such as easy labeling, temperature tolerance, lower cost and recognition of a wide variety of targets. In this study, an aptamer-based PCR method coupled with magnetic immunoseparation was developed to detect S. Typhimurium from ground turkey. Firstly, biotinylated polyclonal anti-S. Typhimurium antibody was immobilized on streptavidin-coated magnetic nanobeads to capture S. Typhimurium. Secondly, the aptamers were added and bound to the surface of S. Typhimurium after blocking the magnetic nanobeads with short ssDNA. Finally, the aptamers were released by heating and amplified by PCR. After optimization, this assay was able to detect 102 CFU/mL of S. Typhimurium in pure culture, and 103 CFU/mL of S. Typhimurium in ground turkey. This study demonstrated the feasibility and application of an aptamer-based PCR method coupled with magnetic immunoseparation for sensitive detection of S. Typhimurium in ground turkey.

      PubDate: 2017-07-01T14:29:50Z
      DOI: 10.1016/j.ab.2017.06.010
      Issue No: Vol. 533 (2017)
  • Measuring kinetic rate constants of multiple-component reactions with
           optical biosensors
    • Authors: David A. Edwards; Ryan M. Evans; Wenbin Li
      Pages: 41 - 47
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): David A. Edwards, Ryan M. Evans, Wenbin Li
      One may measure the kinetic rate constants associated with biochemical reactions using an optical biosensor: an instrument in which ligand molecules are convected through a flow cell over a surface to which receptors are immobilized. If there are multiple reactants, one is faced with the problem of fitting multiple kinetic rate constants to one signal, since data from all of the reacting species is lumped together. Even in the presence of ambiguous data, one may use a series of experiments to accurately determine the rate constants. Moreover, the true set of rate constants may be identified by either postprocessing the signals or adjusting the ligand inflow concentrations.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.06.009
      Issue No: Vol. 533 (2017)
  • A set of enhanced green fluorescent protein concatemers for quantitative
           determination of nuclear localization signal strength
    • Authors: Jennifer Böhm; Ramya Thavaraja; Susanne Giehler; Marcus M. Nalaskowski
      Pages: 48 - 55
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Jennifer Böhm, Ramya Thavaraja, Susanne Giehler, Marcus M. Nalaskowski
      Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.06.015
      Issue No: Vol. 533 (2017)
  • A plate-based histo-blood group antigen binding assay for evaluation of
           human norovirus receptor binding ability
    • Authors: Matthew D. Moore; Lee-Ann Jaykus
      Pages: 56 - 59
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Matthew D. Moore, Lee-Ann Jaykus
      Human norovirus is a leading cause of gastroenteritis worldwide. Although two in vitro cultivation methods have been reported, they cannot provide mechanistic insights into viral inactivation. Receptor-binding assays supplement these assays and give insight into capsid integrity. We present a streamlined version of a receptor-binding assay with minimal time-to-result while maintaining accuracy and high throughput. We validate assay performance for physical and chemical inactivation treatments of a norovirus GII.4 capsid. The assay produces a high positive/negative ratio (25.3 ± 4.9) in <2.5 h and has a limit of detection of 0.1 μg/ml capsid. This method is a valuable additional tool for understanding human norovirus inactivation.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.06.012
      Issue No: Vol. 533 (2017)
  • Development of an immunosensor for quantifying zebrafish vitellogenin
           based on the Octet system
    • Authors: Jun Wang; Jun Wang; Zhenzhong Zhang; Xiaona Zhang; Shaoguo Ru; YiFei Dong
      Pages: 60 - 65
      Abstract: Publication date: 15 September 2017
      Source:Analytical Biochemistry, Volume 533
      Author(s): Jun Wang, Jun Wang, Zhenzhong Zhang, Xiaona Zhang, Shaoguo Ru, YiFei Dong
      Vitellogenin (Vtg) is a sensitive biomarker for environmental estrogens. In this study, an immunosensor for quantifying zebrafish Vtg was developed using the Octet system. First, Protein A sensors were immobilized with purified anti-lipovitellin (Lv) antibody that demonstrated specificity to Vtg. Then, antibody-coated biosensors were immersed into zebrafish Lv standards and diluted samples. The Octet system measured and recorded kinetic parameters between antigens and captured antibody within 5 min. Sample Vtg concentrations were automatically calculated by interpolating relative binding rates observed with each sample and the immobilized anti-Lv antibody into the developed standard curve. The sensor arrays exhibited a wide linear range from 78 to 5000 ng/mL, and the inter-assay coefficient of variation was 0.66–1.97%. Furthermore, the performance of the immunosensor in detecting Vtg was evaluated by quantifying Vtg induction in juvenile zebrafish exposed to 17β-estradiol (E2). Compared with conventional immunoassay techniques, the Vtg immunosensor developed based on the Octet system was much simpler and less time-consuming, allowing rapid Vtg quantification within 15 min. Moreover, Protein A sensors could be reused many times to ensure that the assays have high reproducibility. Therefore, we suggest that immunosensors based on the Octet system are an easily operated detection method for ecotoxicological research.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.07.005
      Issue No: Vol. 533 (2017)
  • Selective fluorescence detection method for selenide and selenol using
    • 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)
  • Measuring fluorescence polarization with a dichrometer
    • Authors: John C. Sutherland
      Pages: 9 - 11
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): John C. Sutherland
      A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.04.001
      Issue No: Vol. 532 (2017)
  • Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide
    • Authors: Suhela Sharif; Jie Shi; Mostafa Bourakba; Rob Ruijtenbeek; Roland J. Pieters
      Pages: 12 - 18
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Suhela Sharif, Jie Shi, Mostafa Bourakba, Rob Ruijtenbeek, Roland J. Pieters
      O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.05.027
      Issue No: Vol. 532 (2017)
  • Fluorescence detection of the transglycosylation activity of amylosucrase
    • Authors: Dong-Ho Seo; Jong-Hyun Jung; Cheon-Seok Park
      Pages: 19 - 25
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Dong-Ho Seo, Jong-Hyun Jung, Cheon-Seok Park
      The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor. The result of HPLC (high performance liquid chromatography) show that the bioconversion of MUG with ASases was successfully accomplished using sucrose and MU. Kinetic studies of ASases were performed to determine kinetic parameter for sucrose and MU. The order of overall performance (k cat /K m ) of transglycosylation activity for MU among DGAS, DGAS-B and NPAS was as follows: DGAS-B (8.1) > DGAS (5.0) > NPAS (0.4). The fluorescence-based transglycosylation assay using MU has a potential to be used as the detection of transglycosylation activity of ASase and to screen novel ASase variants, which may be improved in their transglycosylation activities.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.05.028
      Issue No: Vol. 532 (2017)
  • Fluorescence polarization-based competition binding assay for c-Jun
           N-terminal kinases 1 and 2
    • Authors: Francesco Ansideri; Marcel Dammann; Frank M. Boeckler; Pierre Koch
      Pages: 26 - 28
      Abstract: Publication date: 1 September 2017
      Source:Analytical Biochemistry, Volume 532
      Author(s): Francesco Ansideri, Marcel Dammann, Frank M. Boeckler, Pierre Koch
      In order to evaluate the isoform selectivity of novel inhibitors within the c-Jun N-terminal kinase (JNK) family, a fluorescence polarization-based competition binding assay, previously developed for JNK3, was extended to the other isoforms JNK1 and JNK2. The assay is based on the displacement of a versatile fluorescent pyridinylimidazole-based probe and was validated by testing the precursor of the probe as well as standard JNK inhibitors.

      PubDate: 2017-07-11T06:47:59Z
      DOI: 10.1016/j.ab.2017.05.022
      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
    • 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)
  • 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)
  • 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
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