Hello! I'm doing WB of human brain homogeneates with several antibodies. One of them is strongly reacting with the ladder (Precision Plus Protein Dual Color Standards - Biorad) and is messing with my other lanes.
I'm using BSA 5% for blocking and BSA 3% for antibody incubation, because I'm detecting a phosphorilated protein. For revealing, I'm using LAS4000 machine with standard settings. Only this antibody reacts with the ladder, and the secondary (rabbit) is not reacting with the ladder when using other primary antibodies.
Any idea why this is happening or how to avoid this problem without separating the ladder?
Thank you
That sounds like a non-specific interaction between the MW marker and the primary antibody. Is it the whole ladder or just specific bands? Try loading less marker initially (generally Bio-Rad recommended 7 ul, but you can load less although the markers will be less visible). Another way around could be to mark the position of the marker bands on the membrane with a pencil and then excise the lane containing the marker bands from the membrane before adding your primary antibody (you could also keep the part of the membrane to reassemble with the probed part later when you are imaging the end result).
I would agree with the previous answer. Personally I use 4ul of the same marker as you (Biorad) and it works fine. You could cut the marker after you cut the membrane for incubation. Yet if it's not ruining the final image - why bother
I would definitely be concerned about the non-specific binding. If the antibody is binding to the protein ladder you can't be sure that the antibody isn't binding non-specifically elsewhere too.
However, if you're happy with the specificity of the antibody I'd either do as recommended above, or try using a different protein ladder.
You can try decrease the AB concentration or the time exposition to reduce the unespecific binding.
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