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Development of a rapid and improved two‐dimensional electrophoresis method for the separation of protein lysate
Author(s) -
Kwan Soon Hong,
Ismail Mohd Nazri
Publication year - 2019
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.4686
Subject(s) - isoelectric point , chromatography , silver stain , chemistry , protein purification , lysis , isoelectric focusing , two dimensional gel electrophoresis , polyacrylamide gel electrophoresis , coomassie brilliant blue , immobilized ph gradient , proteomics , electrophoresis , staining , microbiology and biotechnology , biochemistry , biology , enzyme , genetics , gene
Researchers frequently use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D‐PAGE method, which ‘inverts’ the dimension of protein separation of the conventional 2D‐PAGE, is presented in this publication. Protein lysate of Channa striata , a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first‐dimension separation was conducted on a gel‐free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second‐dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D‐PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D‐PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D‐PAGE. Overall, the i2D‐PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.