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Interrogation of an autofluorescence‐based method for protein fingerprinting
Author(s) -
Siddaramaiah Manjunath,
Rao Bola Sadashiva S.,
Joshi Manjunath B.,
Datta Anirbit,
Sandya S.,
Vishnumurthy Vasudha,
Chandra Subhash,
Nayak Subramanya G.,
Satyamoorthy Kapaettu,
Mahato Krishna K.
Publication year - 2018
Publication title -
journal of biophotonics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.877
H-Index - 66
eISSN - 1864-0648
pISSN - 1864-063X
DOI - 10.1002/jbio.201700393
Subject(s) - chemistry , chromatography , fluorescence , isoelectric focusing , sodium dodecyl sulfate , polyacrylamide gel electrophoresis , analytical chemistry (journal) , biochemistry , physics , quantum mechanics , enzyme
In the present study, we have designed a laser‐induced fluorescence (LIF) based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, intrinsic fluorescence spectra of proteins were detected after separation on 1 or 2 dimensional Sodium Dodecyl Sulfate–Tris(2‐carboxyethyl)phosphine (SDS‐TCEP) polyacrylamide gel electrophoresis (PAGE) and the data were analyzed. The MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of protein after 1‐ and 2‐dimensional protein separation. These provided objective parameters of intrinsic fluorescence intensity, emission peak, molecular weight and isoelectric point using a single platform. Further, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise were 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 (309‐356 nm) with pseudo colors allowed the detection of proportion of proteins within the given spectrum. The present methodology doesn't use stains or tags, hence amenable to couple with mass spectroscopic measurements. This technique may have relevance in the field of proteomics that is used for innumerable applications.