I am trying to detect SRC-3. I used primary rabbit antibodies for SRC-3 and beta-actin from Cell Signaling at the suggested dilution of 1:1000 (in freshly made 5% milk). I then used a secondary antibody from a different company. I normally would do 1:10,000 for detection with a Pierce ECL Plus kit, but the company recommended the secondary be used at 1:1000, so this is what I did. I am getting detection of the actin, but not the SRC-3. I cut the membrane at the right MW (~155 kDa), and the membranes for the actin and SRC were both treated in the same manner at the same time points. Do I likely have a problem with my antibody, or could it be a problem with separation of the protein?
Actin is an abundant protein and can be detected at low total protein loading concentrations. Which cells or tissues are you trying to probe? Some proteins maybe present in very low quantities in specific cells. I would increase my loading concentration if that is the case. Also some antibodies work well in 5% BSA. I would try using both 5% milk or 5% BSA . I would also try dissolving the antibodies in TBST or PBS to get the optimum results. Hope this helps.
I've never tested the SRC-3 expression by western blotting.I agree with the answer above, loading more total protein may help you to detect it. In addtion, I think you might notice the difference of the MW between actin and SRC-3. Maybe SRC-3 needs a longer transfer memebrane time.
I totally agree to Jamila and also it would be better if you incubate overnight at 4 degrees with primary antibody. Sometimes it helps me to get my target
Did you see protein with ponceau red? it's not very sensitive, I know but usually you can see faint bands [that at least would tell you if the protein got separated, although that seems not to be the problem as you can see actin. As people mentioned before initial protein concentration is paramount. Also do you use semi-dry or wet transfer? The wet transfer can give better results. Correct me if I am wrong but it is ca 150kDa - that is quite big. I would use wet transfer. You may have not let your gel run long enough??
Or there is no src-3?? Positive control for anti-body?
- Badges
- Science method