In my understanding of WB, band will be appear only where target protein in available, isn't that?
If that, then I should have band in the red marked portion. Why I have lots of scattered band in upper area? What does it mean? Is there my target protein remaining scattered? Or something else?
Please help to understand.
Thanks in advance.
There are a number of reasons. It is possible that your antibody is not very specific, which gives reactions to wrong proteins. It could also be not properly blocked which can give signal where you put the antibodies - possibly the middle of the membrane, giving a band.
Looking at the masses it may be that your protein should be 25 kDa, but the upper bands are in the region of 50 kDa and 75 kDa. This maybe that the majority of your protein is running as dimers, and there is some trimers, but very little monomer. The gel does not appear to have run very well, as the upper bands are a not straight, so it is difficult to say for certain. If this is the case then you need to denature your proteins more strongly - boil for longer, add more SDS and mercaptoethanol to the denaturation buffer.
Hope that helps,
Ellis
There are a number of reasons. It is possible that your antibody is not very specific, which gives reactions to wrong proteins. It could also be not properly blocked which can give signal where you put the antibodies - possibly the middle of the membrane, giving a band.
Looking at the masses it may be that your protein should be 25 kDa, but the upper bands are in the region of 50 kDa and 75 kDa. This maybe that the majority of your protein is running as dimers, and there is some trimers, but very little monomer. The gel does not appear to have run very well, as the upper bands are a not straight, so it is difficult to say for certain. If this is the case then you need to denature your proteins more strongly - boil for longer, add more SDS and mercaptoethanol to the denaturation buffer.
Hope that helps,
Ellis
Dear Mr.Rashid,
Even I have experienced this problem. I think you are using polyclonal antibody. If your antibody is polyclonal then they may produce large amounts of non-specific antibodies which can sometimes give background signal. Polyclonal antibodies not useful for probing specific domains of antigen, because antiserum will usually recognize many domains.
And also check if any problem with running the gel (Make sure there is no leakage or spilling of your protein during loading or handling).
Denature the protein well before running the gel as @Ellis C O'Neill suggested , it looks all protein got collapsed at the middle.
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