International Research Journal of Biotechnology

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Engineering high resolution horizontal native PAGE for proteins: potential in biotechnology analytical applications

Abstract

Kona Madhavinadha Prasad and Vishal Trivedi

Horizontal PAGE is used to analyze negatively and positively charged proteins in a single gel. In the current study, we have developed a simple, user friendly and versatile horizontal PAGE apparatus to run native polyacrylamide gel. We have rigorously tested the ability of the gel system to resolve positively and negatively charged proteins or complex biological samples. The native PAGE resolves proteins based on their charge/mass ratio and a linear relationship was observed between migration (Rf) and the mass / charge ratio of different standard test proteins. Analysis of Bacterial lysate on horizontal native PAGE in 1st dimension and vertical SDS-PAGE in 2nd dimension gives discrete spot pattern on gel. In this current form, the 2-D pattern is not complementary to usual 2-D gel (IEF followed by SDS-PAGE) but it is useful to standardize many factors to observe proteome differences. Besides these analytical applications, designed apparatus can be used to purify native proteins in bulk for biochemical characterization, antibody development, activity assay for pilot studies.

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