Xerographic paper as a transfer medium for western blots: quantification of bovine alpha S1-casein by western blot

We have found that xerographic paper (regular photocopy or laser printer paper) can be used as a transfer medium for protein blots for the immunodetection of low concentrations (a few nanograms) of bovine alpha S1-casein. With paper blotting, we could detect the protein with three times more sensitivity than with polyvinylidene difluoride. Blotting was achieved by transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the methanol-wet paper. The blot was incubated with chicken anti-casein antibodies, sequentially peroxidase-labeled rabbit anti-chicken antibodies, and diaminobenzidine substrate. The blots were directly scanned and the pixel intensity of the band areas was integrated. An analysis of the scanned blots showed that the log of protein concentration was linearly related with the square root of an integrated pixel intensity (r2 greater than 0.96). This linear relationship was observed in a wide range of protein concentration from 5 ng to 15 micrograms. The coefficients of variation were 12.4% for intraassay and 15.7% for interassay. This new analytical procedure can be applied to estimate the concentration of proteins by immunological blotting.