I ran BeWo lysate samples on a polyacrylamide gel in SDS under reducing conditions. Gel was transferred to nitrocellulose membrane and blocked with 5% nonfat milk/PBST. Primary and secondary antibody dilutions were done with the same 5% nonfat milk/PBST prior blotting. Detection was done with BioRad clarity ECL western substrate. I got these weird black spots on the blot instead of seeing any bands (I've validated that the primary and secondary work to give me bands in the past). The far right and left lanes are two different ladders. Anyone have an idea of what can cause these kinds of artifacts on a western blot? I'm not too knowledgeable of western blot troubleshooting.
If you are using a very sensitive substrate, sometimes it generates this kind of artifact.
Yes, I agree with Hamadi. It seems as sensitivity is exceeded whereby artifacts appear and the proteins of the markers are overdyed. To solve this, you could reduce antibody concentrations. But on the other hand, there are not detection of your protein bands. Are you sure your samples have protein? Did you previously perform a protein quantification? If this were not the problem, other possibility could be repeat the assay using non-reducing conditions to avoid a potential lost of epitopes.
Dear Dr Troyer,
Sometimes, the black dots on the blot are from the antibodies binding the blocking agent, you can try to filter your blocking agent to help.
https://www.sinobiological.com/western-blot-troubleshooting-guide-black-dots.html
A speckled or spotty background can be caused by foreign material adhering to the membrane, antibody aggregation, or dirty scanner or cassette surfaces. All are preventable by fastidious handling procedures.
Also once I saw a western that was rinsed with tap water and could see strange spots too.
The above advice from Dr Madhi, and Dr Caballero is good.
Best of Luck!
María Luisa Caballero to answer your question, I did verify that my lysates had protein with microBCA assay prior to running the blot. I did this to standardize input. It is possible that I need to rerun under non-reducing conditions - although as I mentioned I have obtained successful detection with this antibody in the past under reducing conditions. Now that I think about it, it could be an issue with the fact I froze my lysates at -80C prior to use and they have been through 1 or 2 freeze thaw cycles. I will likely retry with fresh lysates and perhaps reducing the antibody concentration. Thanks for the answers all.
This ist most likely due to milkpowder (aggregates) used for blocking. Alternatively, BSA could be used to avoid this effect.
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