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An autofluorescence based method for protein fingerprinting

28 Mar | By Biophotonics.World
An autofluorescence based method for protein fingerprinting
Image source: J. Biophotonics. 2018;11e201700393

Protein purification, differentiation, identification and analysis on a single platform along with added advantage of faster and cost effectiveness is highly desired method in proteomics. Further, stain and tag free visualization of proteins on polyacrylamide gel electrophoresis (PAGE) is another important requirement in proteome analysis to avoid any loss of protein function upon interaction with the added dyes in the process. In order to overcome such limitations, a team of researchers lead by Professor Krishna Kishore Mahato from School of Life Sciences, Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India have designed a laser induced fluorescence based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, a matrix composition was standardized for the effective separation of proteins and detection of the corresponding autofluorescence by replacing Beta mercapto ethanol commonly used for reducing protein disulfide bonds, with Tris corboxy ethyl phosphine (TCEP). Autofluorescence signatures of proteins were detected after separation on one and two dimensional (1D and 2D) Sodium Dodecyl Sulfate (SDS)-TCEP-PAGE by shining them with 281 nm laser lights and targeting their tyrosine and tryptophan moieties. Subsequently, a MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of proteins after 1D and 2D protein separation. 

Dr. Mahato says, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise are not identifiable by conventional staining, imaging and tagging methods. Categorization of the proteins based on the presence or absence of tyrosine or tryptophan residues and assigning the corresponding emission peaks (309-356nm) with pseudo colors allowed the detection of a proportion of proteins within the given spectrum. The present methodology does not use stains or tags, and hence amenable to couple with mass spectroscopic measurements. 

For the first time, the methodology is standardized to record the autofluorescence spectral properties of proteins from PAGE without interference from the chemicals used in the PAGE gel preparation. This development opens up a door for in gel measurement of complex autofluorescence properties of proteins such as fluorescence decay and lifetimes said Manjunath Siddaramaiah, first author of the study.  

The findings are now published in the peer-reviewed journal, “Journal of Biophotonics”. After successful development of the method the team is now involved in the miniaturization of the setup for its commercial applications.


Author: Krishna Mahato


Related journal article: Interrogation of an autofluorescence-based method for protein fingerprintin(J. Biophotonics. 2018;11e201700393)

Area of application: Cell Biology

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