A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics

Talanta
Chen-Gang GuoCheng-Xi Cao

Abstract

Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots co...Continue Reading

References

Mar 14, 2003·Nature·Ruedi Aebersold, Matthias Mann
Jun 26, 2007·Nucleic Acids Research·Leonid ZamdborgNeil L Kelleher

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Citations

Jun 4, 2019·Analytical and Bioanalytical Chemistry·Fátima M SantosLuís A Passarinha
Mar 2, 2017·Proteomes·Melissa R Pergande, Stephanie M Cologna
Sep 17, 2019·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Shah SaudChengxi Cao

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