I am doing the western blot with ECL method in mouse system. I transfer the proteins to PVDF membrane with an efficiency about 90-95% confirmed by both Commassie blue and Ponceau-S staining. The secondary antibody is cojucated with AP. I get substrates from Molecular probe. With the help of AP the substrates can emit chemilluminescence and detected by the film from Amersham (exposure time either 60 or 20 seconds). The question is that I always get high background even with secondary antibody only. The concentrations for the 2nd antibody are 1:5,000, 1:10,000, and 1:15,000 in 1xTBS-T, respectively. I wonder if anyone out there use more diluted 2nd antibody and less exposure time or good method to reduce background?
Thanks in advance,
Edited by alex_osu3, 30 April 2004 - 01:39 PM.