I'm looking for the cause of black dots within a Western blot band (see attached image). I don't see these dots in the background, but they show up where my protein is supposed to be.
The primary antibody is new and I made sure to spin down the secondary before use (usually doesn't produce bands like this). This membrane was previously stained and was stripped prior to current blot. Any ideas?
Hi Joe! If I got correctly, you detected these dots where the protein was expected to be after one stripping step. Maybe your stripping was to aggressive and you detected the rest of expected protein. Maybe you should assess the initial protein concentration or the stripping solution.
Good luck
Samyr
It could be incomplete denaturation of the samples causing uneven lane loading. Did you notice anything odd during separation (e.g. the dye front migrating as a series of spots rather than a flattened line)? Likewise, do you have PonceauS or Coomassie S images of the gel/membrance? Finally, do other primary antibodies cause the same sort of banding pattern?
Samyr Kenno Yes I was wondering whether this was a problem with stripping the membrane. I used a commercial reagent for the recommended time, so not sure if this staining pattern is due to that.
Brian Bower The first time the membrane was stained, the target protein and loading control looked fine - fairly straight and even. I haven't seen this banding pattern in any of my other primaries.
Did you checked membrane by PonceauS before an antibody treatment?
Maybe you have to check if % of the gel, transfer buffer and transfer conditions are apoppriate, especially if you have "big" protein.
Might be bad transfer to the membrane..or some of the bound protein came off. Best to test the ab on a control and try again. Try Ponceaus S as suggested as well but other proteins might have the same Kd.
Regards,
J-P.
I think above suggestions are likely explanations to your dilemma. One thing could still be that you detection antibody has aggregates. Spin down the conjugate 1 min 16 rcf and then make the dilution for the western, might help
Best
Juha
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