I got high background noise on my blots after detection. Can it be a problem due to protocol I'm using for western blot?
After transfer I'm staining with Ponceaus S, i'm washing a bit with distilled water, taking photo. Then blocking in TBST with 3% milk (mixed before on magnetic stirrer). I flood the gutter with blots and incubate around 1h in RT. Then i'm taking 5ml of TBST with 3% milk with (antibodies dissolved in it) into falcon and incubate blots over night. Next day im washing blots 3x15minutes with 5ml of TBST and then incubating with 5ml of TBST with secondary antibodies (1h). After that i'm washing once again 3x15 minutes with 5ml of TBST. After that detection.
You can try increasing your milk to 5%, and using milk in the TBS with your secondary. CST normally recommends 5% milk with their antibodies and the secondary and I've had pretty nice results.
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