Western immunoblotting: temperature-dependent reduction in background staining

The effect of incubation temperature on the background staining of Western blots with monoclonal antibodies to a human milk protein, alpha-lactalbumin (Mr 14,500), is presented. Human milk proteins were electrophoretically separated and transferred to nitrocellulose membranes which were then blocked with bovine serum albumin, "BLOTTO", casein, or Tween 20. They were subsequently incubated with mouse monoclonal antibody to human alpha-lactalbumin, biotinylated anti-mouse antibody, strepavidin-biotinylated horseradish peroxidase complexes and a substrate containing diaminobenzidine and nickel chloride. Reduction of incubation temperature from 37 degrees C to 22 degrees C and 4 degrees C was found to decrease the extent of non-specific background staining independent of the type of blocking reagent used. Good specific staining with minimal background was found using 0.1% Tween 20 in phosphate-buffered saline, pH 7.2, as blocking agent and incubation temperatures of 4 degrees C.