I have done western blots for several months with LNCaP (prostate cancer cells) cells and want to detect antibodies between 50-70kDa. My problem is that there is an unspecific band in each of my blots which appears after several seconds and is quite strong. Unfortunately this band often covers the bands that I want to detect which is really frustrating.
This is the protocol I am following:
1. I run the proteins (15µg/pocket) in SDS Page Gels which we produce by ourselves.
2. After that I transfer the proteins for 30mins to the membrane
3. 5min washing with TBST
4. 1h of blocking either with 5% milk or 5% BSA in TBST
5. Primary antibody (1:500-10.000) overnight at 4 degrees.
6. 3x5min washing with TBST
7. Secondary antibody (rabbit or mouse) 5µl with StrepTactin 2µg in 10ml TBST
8. 3xmin washing with TBST
9. Detection of the bands
Down below you can see some pictures of this unspecifc band. I would be really helpful for me to get rid of this unspecific band as it keeps me from detecting all my antibodies...
Do you mean recombinant antibody in your cell constructions? Otherwise, I guess that the band at 55-60 kDa, which appears to associate with another smaller band at 22-25 kDa in the attached figures represent heavy and light chains of one of the immuniglobulin(s), likely overproduced in LnCAP cells. The size variability can be related to an extensive post post-translational modification or running conditions. I suggest you to perform the depletion of a putative Ig(s) by pre-cleaning (no immunoprecipitation!) with Protein A or, better, with Protein G-conjugated agarose from Santa Cruz, Bio-Rad, etc. Run the pre-cleaned and non pre-cleaned samples with control IgG in the same gel. I would be glad to hear that you could get correct results.
https://www.researchgate.net/post/On_my_western_blot_membrane_i_receive_nonspecific_bands
Hi Melina Andresen . The most common fixes are to reduce the amount of 1o and/or 2o antibodies or try antibodies from other sources.
Vehary Sakanyan
Thank you very much for your answer! Could you send me a link to the Protein G from Bio Rad? I can't really find it on their website.
Please, find the reference for Protein G PLUS-Agarose: sc-2002 from SANTA CRUZ BIOTECHNOLOGY, INC. Preclear samples is explained in the Procedure, the line 12th "Preclear lysate (optional step)... Normally, this step is enough to eliminate immunoglobulins that likely mask your 50-70 kDa protein. You can use smaller volumes of lysates.
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