Hi, I am using monoclonal antibody against B-actin but I got multiple bands in that blot. I tried 1:100, 1:500, 1:1000, 1:2000 and 1:5000 primary antibody concentrations, but still same thing happens. I am using secondary antibody 1:5000 and 1:10000 conc. I am also used 5% BSA and 5% skimmed milk for blocking and for antibody incubation. I also tried by putting blot for overnight blocking. I am trying incubation of primary antibody for overnight at 4 degree Celsius and at 37 for 2 hrs, still I got 4 bands instead of band of interest. I also tried multiple washing loke for 5-7 times. Please give me any suggestions.
Sometimes it is the primary antibody which is not good! I would try with another one...
From which source is your protein, which antibody are you using, can you post a picture?
And have a look at the answers to similar questions at RG: https://www.researchgate.net/post/Does_anyone_have_experience_with_multiple_b-actin_bands_in_western
https://www.researchgate.net/post/Can_someone_share_their_experience_on_multiple_b-actin_bands_in_western
If your primary antibody is binding multiple proteins there's nothing you can do except get another primary antibody. If the signal is independent if the primary antibody (e.g. you have immunoglobulin in your samples recognized by the secondary) that's another story.
Sabine Strehl @ the arrows showing non specific bands in blots in image attached. i am using rat brain tissue as a source of protein.Antibody I am using is from Sigma (SAB1403520) which is monoclonal.
The issue would seem to be with either your antibody or your protein samples. Is the antibody proprietary or is it one your lab had made? If the latter, it might be useful to see if you can get a known standard for beta-actin and test the antibody on that, to see if you keep getting the multiple bands. It is also possible that your protein sample may have degraded. If you keep it at 4 degrees long-term without some preservation agent, or if you freeze and thaw a sample repeatedly, the quality of the sample goes down considerably.
with antibody as simple and well expressed as beta actin, you really should only see 1 band. I use the antibody from sigma product # A5441 at a dilution of 1:2500 and it works perfectly with 1 band. I am not sure what antibody company you are using, but like others have stated it sounds like the antibody that you are using is not specific.
Hi! If you are sure of the specificity of your antibody then the problem can be your samples. Did you add proteinase inhibitors?
I would consider these questions: Are you using sufficient protease inhibitors? How much protein are you loading per lane? And, have you probed for any other proteins in this sample set, or on this blot? If so, what were the results?
Try to use new cell lysate, sometimes when you store the lysate at -20, if it s not enough to stop their degradation
Also try higher dilution, for me i am using EF1 alpha and the recommended dilution is 1:1000, but i got multiple bands, so now i am using 1:10000 and it works great
I have the same challenge for just one particular protein blot here. My samples were used to detect the expression of this protein but I noticed double bands. I used the marker with the knowledge of the molecular weight range to make my choice. What I thought was that if the blot could give this result for only a protein and not for others, then the probability stands to be certain that the antibody is the one at fault and not my protocol or technique or sample preparation. Maybe I was wrong, but that is the way I think it.
I had somewhat similar kind of problems with actin. Lysing your samples on ice and transferring on ice could help. Also the major problem could be with your primary antibody. Try to use primary antibody from a different company.
Could there be any protein degradation in your sample? That would result in multiple bands. Another option is lack of specificity in the antibody.
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