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Proceedings Paper

Autofluorescence based visualization of proteins from unstained native-PAGE
Author(s): Manjunath S.; Bola Sadashiva Satish Rao; Kapaettu Satyamoorthy; Krishna Kishore Mahato
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Paper Abstract

Proteins are the most diverse and functionally active biomolecules in the living system. In order to understand their diversity and dynamic functionality, visualization in native form without altering structural and functional properties during the separation from the complex mixtures is very much essential. In the present study, a sensitive methodology for optimal visualization of unstained or untagged proteins in native poly-acrylamide gel electrophoresis (N-PAGE) has been developed where, concentration of the acrylamide and bis-acrylamide mixture, Percentage of the gel, fixing of the N-PAGE by methanol: acetic acid: water and washing of the gel in the mili-Q water has been optimized for highest sensitivity using laser induced autofluorescence. The outcome with bovine serum albumin (BSA) in PAGE was found to be highest at acrylamide and bis-acrylamide concentrations of 29.2 and 0.8 respectively in 12% N-PAGE. After the electrophoresis run, washing of the N-PAGE immediately with miliQ water for 12 times and eliminating the methanol: acetic acid: water, fixing of the N-PAGE yielded better sensitivity of visualization. Using the above methodology 25ng of BSA protein band in PAGE was clearly identified by the technique. The currently used staining techniques for the visualization of proteins are coomassie brilliant blue and silver staining, have the sensitivity of 100ng and 5ng respectively. The current methodology was found to be more sensitive as compared to coomassie staining and less sensitive compared to silver staining respectively. The added advantage of this methodology is the faster visualization of proteins without altering their structure and functional properties.

Paper Details

Date Published: 9 March 2015
PDF: 6 pages
Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 933113 (9 March 2015); doi: 10.1117/12.2079302
Show Author Affiliations
Manjunath S., Manipal Univ. (India)
Bola Sadashiva Satish Rao, Manipal Univ. (India)
Kapaettu Satyamoorthy, Manipal Univ. (India)
Krishna Kishore Mahato, Manipal Univ. (India)

Published in SPIE Proceedings Vol. 9331:
Single Molecule Spectroscopy and Superresolution Imaging VIII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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