Modified diffusion blotting for rapid and efficient protein transfer with PhastSystem

Abstract

An easy‐to‐perform and efficient blotting method was developed for the transfer of proteins separated in PhastGel media. The method was evaluated by comparing the blotting results of low molecular weight marker proteins at different times. ¹⁴C‐Labelled proteins were used for the assessment of the transfer efficiency. Highly efficient transfer was achieved within 1 h of blotting and the proteins were almost completely eluted from the gel in 2 h. The gel remained on its solid plastic backing, which is not the case when using an electrophoretic transfer method. As little as 1.25 ng of electrophoretically separated protein were enzymatically detected on the blot when applying blot‐biotinylation. The transfer was performed at low temperature (4°C), which – in contrast to thermoblotting – additionally offers the chance to blot thermolabile proteins. These superior features, together with the method's versatility and low cost, mark an interesting alternative to the previously recommended blotting procedures after PhastSystem electrophoresis.