Hi all, I cut the western blot membrane and incubated it with two different antibodies according to the molecular weight. The result showed that one membrane is clean and has no non-specific band, but the other membrane shows a significant black signal. I thought it was a speck of dirt, so I filtered and freshly prepared all the reagents—however, the same black signal was still in the blot, and the shape was the same. Have anyone else suffered from the same issue? How do you fix this problem? Many thanks!
Hi,
what kind of blocking solution did you use? I´ve got best results with non-protein based blocking solution - check: https://www.thermofisher.com/order/catalog/product/37584.
Than, try to wash your membrane at least three times with buffer before you will visualize proteins and than dry the membrane pretty well using filtration paper. Let me know please, if it helped.
BR,
Maroš
In addition to Maroš Huličiak 's suggestion, make sure that the antibody solution is well distributed on your membrane during the incubation step on the shaker. We have noticed in our lab that when we use lower or larger volumes of antibody solution, we tend to have membranes that look the same as yours. It is also important to properly wash your membranes after each antibody staining (3 times 10 minutes in TBST 1X).
Goodluck!
You imaged two piece of membranes exposed to two different antibodies in parallel and used both piece of membrane from the same roll. One membrane is clean and with no non-specific band, but the other membrane is showing big black signal. If correct, then there is almost no issue with your procedure and the membrane since one of the blots is clean with no background.
Issue could be with your antibodies (priAb & secAb) that were used to detect the protein of interest on the blot of your concern.
1) Check whether the priAb and secAb are working fine (not degraded) and dilutions are as per company datasheet for both antibodies.
2) Check whether membrane was correctly & equally exposed in primAb solution during incubation.
3) Use of less amount of priAb solution may allow the membrane to trap air on the bottom side and solution may get stuck to some peripheral area of the membrane leading to generation of such such blot.
4) Check whether your protein of interest is phosphoprotein and you are using milk protein in blocking buffer. If yes, replace the blocking buffer as suggest above by Maroš Huličiak.
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