This book presents modern and classic analytical approaches that are crucial for the biochemical and functional characterization of the archetypal protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The distinguishing feature of the book is that it covers, in addition to other methods, some of the uncommon but valuable techniques as well. For example, in-gel visualization of enzyme activity, immunoblotting protocols for native (non-denatured) proteins, and proteins resolved by pH-gradient [IEF-isoelectrofocusing], etc. These expedient methods are relevant and vital for the verification of biochemical properties of GAPDH, or similar protein of interest.

This work outlines detailed protocols that are essential to investigate classical (cellular) and recently reported extracellular (secretory) isoforms of GAPDH. Precisely, the book covers techniques pertinent to enzymatic and non-enzymatic analysis of GAPDH that include, but not limited to, electrophoretic mobility shift assay (EMSA), two-dimensional (2D)-immunoblotting, immunofluorescence/confocal microscopy, mass spectrometry, ion-exchange and affinity chromatography.

Readers will discover the importance of the experimental methods described in the book as they relate to the evaluation of the role and significance of GAPDH. Furthermore, majority of the methods described in the book have also been validated in the author's laboratory, besides other research groups worldwide, underlining the repeatability and reproducibility of the protocols. Each method begins with an abstract and a brief background emphasizing its application and relevance. This will enable the readers to determine the choice of experimental design according to their research objectives.

The book explains the methods systematically with ample illustrations to facilitate quick and easy comprehension of the practical knowledge. Although the book is focused on GAPDH, many of the protocols may be adopted to other proteins or enzymes with minimal modifications. Noteworthy, it is unequivocally established that GAPDH is a multifunctional protein involved in several cellular processes of health & disease conditions. Hence, this book will be a valuable practical guide for young researchers, scientists and clinician-scientists.

Is a unique compendium that offers a complete solution of protocols on GAPDH protein analysis for researchers at all levels including undergraduates, graduates,postgraduates and research scientists in life science /general and applied biology Outlines exceptional methods to analyze enzymic proteins (e.g. GAPDH) under native, non-denatured condition by immunoblotting Illustrates both qualitative and quantitative analysis of GAPDH as an enzyme and as a non-enzymic protein Encompasses methods for the analysis of recently discovered isoform, secretory-GAPDH Offers notes/tips that are essential for the successful completion of protocols Provides nice illustrations to support the text

Autorentext

Shanmugasundaram Ganapathy-Kanniappan holds a Ph.D. degree from the University of Madras, India. In 2006, he joined the Department of Radiology, Johns Hopkins University School of Medicine, as a junior faculty member and was quickly promoted to assistant professor. Before joining the Johns Hopkins, he underwent postdoctoral training in premier institutes such as the National Institute of Immunology (NII, New Delhi, India) and the University of California at Los Angeles (UCLA). His current research interests include metabolic alteration in human pathophysiology and the identification of molecular targets for therapeutic intervention. He has published over 50 articles including original reports, invited reviews, editorials, book chapters, and books (https://www.ncbi.nlm.nih.gov/myncbi/1xKsymd-il85x/bibliography/public/). He is an inventor and coinventor of several patents and a recipient of prestigious fellowships and research awards including funding from private and federal sponsors. He is on the editorial board of peer-reviewed journals and currently serves the journal, Frontiers in Oncology as an associate editor for the section, Cancer Molecular Targets and Therapeutics.

Inhalt

Introduction.- Analysis of GAPDH enzyme activity: A quantitative and qualitative approach.- Analysis of GAPDH - Native protein.- Analysis of GAPDH under Denaturing Conditions.- Analysis of GAPDH and Protein Interaction.- Analysis of GAPDH and Nucleic acid Interaction.- Analysis of Subcellular and Extracellular GAPDH.- Analysis of GAPDH posttranslational modifications.- Analysis of GAPDH by Mass Spectrometry.- Purification of GAPDH.