non-specific bands - (May/01/2010 )

Hi all,

I am just wondering if anyone knows of how to tell if you have non specific bands on your blot due to lack of specificity of the antibodies. I originally had antibody from santa cruz but then was told that the antibody for the particular transcription factor I'm looking at might be non specific due to the source of the antibody. So then I bought another one from Abcam, rabbit monoclonal antibody recognizing carboxyterminal end of the transcription factor . I have run western for cytoplasmic extract of cultured cells with the antibody from abcam and have a band at 100 kDa. The issue is that the expected molecular weight is about 57 kDa. A reviewer of the antibody from Abcam also reported a band at 100 kDa, which is generally the ubiquitinated form of the transcription factor in the cytoplasm. Nevertheless, when I look at pictures from other antibodies in Abcam (for the same transcription factor but just recognizing the whole thing or amino ends, and it's mostly polyclonal), it looks like there should be two bands, one at 57 kDa and one at 100 kDa, suggesting the presence of the non-ubiquitinated protein and ubiquitinated protein, respectively. The picture is similar to what I saw in Santa cruz website for the transcription factor. So my question is.. how would I be sure that the two bands that I might be seeing with my Santa cruz antibody (haven't tried them yet), will be specific enough to my transcription factor? Also, I wonder what difference would it make if the antibody is monoclonal or polyclonal?

oh and another basic question is why would a certain source of antibody have a concentration of the antibody and the one that I purchased from abcam doesn't? it says:
"Concentration not applicable. It is not possible to determine the concentration of this antibody, since tissue culture supernatant contains proteins besides the antibody of interest.

Supernatant is the fluid resulting from centrifugation of hybridomas in tissue culture that are secreting specific monoclonal antibodies. Supernatants therefore typically contain culture medium and 5-10% fetal calf serum. Antibody concentration is only relevant for purified antibodies."

Thank you for any response I can get!

zienpiggie on May 2 2010, 02:26 AM said:

why dont u use a negative control and find out for any nonspecificity. Also sumtimes the secondary are non-specific so try to develop a blot using only the secondary antobody to see if it gives any non-specific bands!!

The best way to determine specificity of an antibody is to do a peptide competition - run a sample (that gives you multiple bands) in a couple of lanes of the same gel, blot onto a membrane and then cut the membrane into two bits. On one half probe with the antibody as you would normally. On the other half, pre-incubate the antibody with an excess of peptide that contains the epitope of the antibody, then use the "competed" antibody to probe the membrane. detect as usual for both halves and compare. Specific bands should disappear from the competed membrane, bands that are still present are non-specific.

You can often buy peptides against which antibodies are raised from the company that manufactures the antibody. If they don't have one available, demand the epitope (and hope that it isn't full length sequence).

Hello,

I also had a problem with unspecific bands. When I tried to detect NF-kB p65 I got the 65 kDa bands but also unexpected bands of about 150 kDa. Does anyone know what that may be? This blot was detected after a stripping but the "old" protein was also 65 kDa and had been (apparently) completely removed. So I don't think that should be the problem.

Thanks

Thanks guyz. The synthetic peptide idea sounds pretty good.