Multistrip western blotting to increase quantitative data output

Abstract

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

Fig. 1. Gel loading strategies when the number of samples exceeds the number of gel wells

Fig. 2. The identification and cutting of protein migration zones in Multistrip Western blotting procedure

M, prestained protein molecular weight marker; BDF, blue dye front; H, the distance from BDF to the center of particular marker band. Scissor symbols indicate the cutting lines, which would separate the entire gel into nine strips. Protein migration zones are enumerated by numbers in circles (modified from ref. 19 with permission from WILEY-VCH Verlag GmbH & Co. KG aA).

Fig. 3. Example of single gel cutting into five (left panel) or four (right panel) strips containing distinct protein zones

The strips will be subsequently transferred onto assembling filter papers together with similar strips derived from other gels.

Fig. 4. Assembly of multiple gel strips onto assembling filter paper

A. SDS-PAGE of six gels (A through F) was performed. Five strips were cut out of each gel and combined onto appropriate assembling filter papers (AFP, #1 through #6). B. SDS-PAGE of three gels (A through C) was performed. Four strips were cut out of each gel and combined onto two assembling filter papers (AFP #1 and #2).