Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel

• Published in: Proteomics (Weinheim) Vol. 14; no. 21-22; pp. 2437 - 2442
• Main Authors: Kinoshita-Kikuta, Emiko, Kinoshita, Eiji, Matsuda, Ayumi, Koike, Tohru
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01-11-2014; Wiley Subscription Services, Inc
• Subjects: Blotting, Western - methods; Electrophoresis, Polyacrylamide Gel - methods; HeLa Cells; Humans; Phos-tag SDS-PAGE; Phosphoprotein; Phosphoproteins - analysis; Phosphoproteins - isolation & purification; Phosphorylation; Pyridines - chemistry; Semidry blotting; Technology; Western blotting; Wet-tank blotting; Phos-tag SDS-PAGE; Phosphoprotein; Technology; Wet-tank blotting; Western blotting; Semidry blotting
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.201400380

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.