I recently started to do western blots and I'm currently testing several antibodies against MAPKs that none im my lab used before, so I have to find proper conditions to use them for my samples.
Some antibodies gave nice blots, others took a bit tweaking with blocking and primary antibody conditions,but with others I have that problem that when I take photo (using Amersham AI600 imager) i get black or blotchy photo. That is photo captured in mode with colorimetric marker. Whereas, .tif images of the same blot (it consists of 2 layers, picture of detected proteins and of standard), sometimes have only faint bands, without black blotches, and sometimes just grey blotches.
I played with conditions, tried changing blocking buffer 5% BSA to 5% skimmed milk, for 1h or overnight, antibodies in 1% BSA or skimmed milk overnight at 4C or 2h at RT. Even tried primary antibody diluted without blocking agens or in 5% BSA or skimmed milk. Tried diluting or concentrating primary antibody and diluting secondary but without success. I also tried different concentrations of protein, 20-40 ug per well.
I even briefly thought it is some problem with capturing of images or chemiluminescence detection reagens (which we make ourselves in lab), but then I wouldn't get nice photos for any of the antibodies.
Does anyone have a suggestion what else could I do to solve my problem?
Images are attached.