I am currently doing western blot for p-Akt. Below is my protocol:
-- Blocking = RT, 2 hours with 5% BSA
-- Primary antibody = p-Akt (1:2000), overnight, RT
-- Secondary antibody = Anti rabbit with HRP (1:3000), 1 hour 30 minutes, RT
-- Exposure = LAS Image 500
Result = black blot with no bands
Any suggestions?
Your protocol ressembles the one suggested by Abcam for phospho antibody, which is a good thing. Maybe you can try decreasing the concentration of the secondary antibody to 1/10000 or lower. Maybe add also the secondary antibody in the blocking plus 0.1% tween instead of TBST.
Uniform high background can result from a variety of reasons:
1. High Concentration of Antibodies
2.Insufficient Blocking
3.Interference from incompatible blocking agents
4. Insufficient washing
5. Membrane type (PVDF or nitrocellulose)
6. Bacterial contamination of buffers
7. Overexposure of membrane
try to play with this and see what will help you
BSA interacts with many proteins easily (may be the end of the last century, I read a paper reporting it. I'm sorry I can not show it now.) Therefore it is better to use skim milk or other blocking kit and wash sufficiently.
i am using ImageQuant LAS 500 to detect the band..
if I included the colorimetric marker, the blot was very dark.
However, if I just did for chemiluminescence, the blot was not dark, but no band
I have no idea what is the problem
BSA is recommanded over milk for Phospho antibody because casein present in milk is a phospho protein and therefore using milk as blocking can produce some background in this case.
I imagine that if your antibody is very specific for a specific phosphorylated peptide, using milk should be less of a problem. I agree with the comment above, that BSA generate more often than milk the problem described in the question. The troubleshooting list of Petra above is also very useful. Membrane and secondary antibodies are the first thing to try. Exposure is also key: WB with strong signal should be exposed to film for a little as 10 seconds. If required to go below, you should reduce the amound of antibodies used. When I had too much signal, I often saved a day of work by putting two films over my membrane to reduce by filtering the signal crossing to the second film.
I have noticed some thing in your protocol that if you want to incubate with primary antibody overnight, you need to put it in the fridge not at room temperature!!. we usually incubate overnight if we see faint bands, is this why you incubate overnight?. Other than that, I agree with most of the comments mentioned in this dialogue.
In case of high concentration of secondary antibody, what can someone do for correction and still retain the membrane
- Badges
- Science method