Hello, I am working with the HMC3 cell line and have been having issues with western blot, particularly when blotting for caspase-4. I am getting incredible amount of non-specific bindings in my western blots and I don't know what else I can do to fix it at this point. We have a standard protocol in our lab and people have had beautiful blots with the caspase-4 antibody we are using. We use 5% milk+TBST for blocking and it works for other people. I have tried longer blocking (e.g. 2hrs, overnight blocking) and it didn't work. I have tried lower primary Ab concentration (1:5000 instead of 1:2000 which is what other people use in my lab) and it didn't work. The dilution factor for secondary Ab is 1:5000 which I don't think it's too high. We wash the membranes 3 x 15 min after primary and secondary incubation respectively, and I have increased the washing time up to 3 x 30 min and the non-specific bindings are still there.
I was thinking it may be a problem with the cell line I am working with, but I see similar degree of non-specific bindings when I borrowed other people's samples. I don't know what I am doing wrong here to cause the degree of non-specific bindings I am seeing... Also, when I blotted the same samples for other proteins e.g. GSDMD, caspase-3 and 7, they are much cleaner. Therefore, I still would like to ask whether anyone's working on the same cell line and is having similar issues?
Here are some suggestions;
- Change your membrane material (PVDF to nitrocellulose or vice versa)
- Test the BSA ingredient in your blocking buffer instead of non-fat dry milk powder
- Test the commercially available detergent-based blocking buffer formulations. You may also prepare one in-house using Triton X
- Try to refresh with a new one or change the brand of your primary antibody
good luck
Hello Yunjia Chang
Other proteins like GSDMD, caspase-3 and 7 are much cleaner using the same samples which means that there is no problem with the cell line. I feel you should check the primary antibody. How sure are you that caspase-4 antibody is in good condition?
If the primary antibody is degraded, it can lead to non-specific bands in a Western Blot because the degradation process can alter the antibody's structure, potentially exposing new binding sites that lead to off-target interactions. These altered binding sites can lead to the antibody binding to proteins other than the target protein of interest, resulting in non-specific bands on the Western Blot.
Also, make sure that your sample is properly prepared, and you are using sufficient protease inhibitors to prevent degradation of proteins, which can affect antibody binding.
Best,
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