You can try to reduce secondary antibody.

Thanks...any other suggestions

increase the number of washing steps and use more Tween in PBS

Hi Sanjaya (nice to see you again), Santa Cruz is really a bad company for antibodies. I try to avoid ordering an antibody from them even they are the cheapest. Nevertheless, you can try to increase the signal to noise ratio by centrifuge a lower amount of your stock (roughly 50 µL) in an ultracentrifuge at 30-40,000 rpm for 1-2 hrs and then I guess you will see a small pellet which are immunoglobuline complexes and are the reason for your high background. Take the supernatant (leave a little bit on top of the pellet to make sure that you are not pipetting your complexes over) and transfer it into a new tube and use this a little bit less diluted then before for your western blot detection. It helped a lot when I did it. On the other hand, Bethyl lab, Abcam and Millipore are great companies well known for their good antibodies in most of the cases. Good luck and see you soon again ;-), Nicole

@Esen thanks....yeah will try that

@Nicole actually I was trying to get the antibodies for phosphorylation studies and total Bcl-2; antibodies for phosphorylation studies that have been mentioned in the NIH website is Santa Cruz...I myself had hunch that it might not work well with its reputation so far, but then again since other companies antibodies did not have a reference of being used in any other experiments the p87-Bcl2 one..I had to get theirs, however the phosphorylated form I am still to use. Hope it works, regardless of the ghost bands... I will try ultracentrifuging the antibody and use it as you mentioned. Thanks again.