I perform western blot according to LiCOR odyssey protocol. When I scan my membrane on odyssey CLx, I get dark blobs or bands at around 65 KDa. It seems to show up when I use some antibodies but with other antibodies on the same samples, I don't get these dark bands. Has anyone faced the same issue? Any insights would help.
Hi Deepak! Do you use the same protocol for all the different antibodies? It might be that the antibody dilution is too high, or the incubation time was too long. An image of the blot will help though.
Hi Deepak, is it possible that you visualize heavy chain antibodies seperated on your gel? Did you separate proteins from a source that may contain antibodies or did you perform an immune precipitation?
What kind of samples are you separating? If you are using cell culture supernatants containing fetal bovine serum, you might very likely have albumin contamination, which is around 66 kd and often gives very high aspecific binding to antibodies.
hi deepak,
I second Marco suggestion about BSA contamination, specially if your concentration of secondary antibodies is too high. At high concentrations secondary antibodies precipitate unspecifically over protein clumps.
The major challenge with this type of issue is the dilution of the primary antibody. I think, with your explanation different antibody might have different dilution factor. You may have to pay attention to your dilution factor. Some antibodies may have 1microlitre:500microlitre while some may have 1:1000 and if you are using a control like beta-actin, I always use 1:2000 and it gives clean beautiful bands.
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