Biomedical chromatography : BMC | 2019
Development of a rapid and improved two-dimensional electrophoresis method (i2D-PAGE) for the separation of protein lysate.
Abstract
Researchers have widely utilised the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis by proteomics approach. The i2D-PAGE, 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 molecular weights in the first dimension and followed by isoelectric point (pI) 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. Second dimension separation features isoelectric focusing, which further separated the proteins within the same fraction according to their respective pI. Advantages of the i2D-PAGE include better visualisation on the isolated protein, easy identification on protein isoforms, shorter running time, customised and reproducible. Erythropoietin (EPO) standard was applied on the i2D-PAGE to show its effectiveness on 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 to be applied in the proteomics research.