After performing western blot (oxyblot), I do see my bands, but I also get patchy background. and I notice it is usually the spots where I put my ECL on first then gets the background. I have no idea what is happening. Any advice to remove the background? Please advice. Cheers
Zi
Hey, would you mind to upload the image of your developed film?
Please describe the steps you did eg. blocking reagents... so that we can help to troubleshoot.
Without seeing your blot there are a few things you can do:
if its patchy it does not sound like a direct problem with the antibodies. If they have been mixed properly you would expect a uniform background if there is interference here
1. Washing steps are inconsistent: insufficient mixing/coverage. Increase stringency of wash solution (ie. increase Tween 20)
2. incubation with antibodies is inconsistent: membrane not fully covered with solution, insufficient mixing.
3. Blocking solution is causing background, Try different blockers
4. Handling of blot without gloves/blot contamination. Clean apparatus and mixing vessels before use.
5. Blocker not fully solubilised or insufficient blocking
6. Pieces of gel attached to membrane after transfer maybe the cause of small patches of background
7. Membrane allowed to dry out after addition of antibodies
8. Dirty Hypercassette (if using manual)
bw
Ian
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