Hi everyone. I have just used for the first time a NovusBio GSTA2 antibody (cat. no. H0002939-D01P). As reported in the figure , three different samples were used in our preliminary Western blotting experiment. Surpristingly, only one of our samples (mouse ovary), expressed the "right" band at approx. 25 KDa. The other two samples (both from cells) did not show any 25 KDa band. However, all our samples seem to present strong immuno-signals slightly above and below 63 KDa. Interestingly, the only sample that showed the 25 KDa have weaker signals around 63 KDa.
May it be possibile that the extrabands with higher molecular weights could be complexed/immature forms of the protein of interest?
Has anybody encountered such a problem with cell lysates?
Thanks so much in advance
Stefano Falone, Ph.D. Department of Life, Health and Environmental Sciences Section of Biological and Biotechnological Sciences University of L'Aquila 67100 L'Aquila (AQ) - Italy Phone +39 0 862433260 Email [email protected]
You can try to use DTT instead of B mercapto, but your denaturation conditions are already strong, it can be non specific binding at all.
There are many tricks to try:
-Changing your lysis buffer with stronger one and more SDS
-Try different membrane saturation methods/time.
-Try another antibody
-Transfert shorter time to prefer the lower weight proteins
-Cut your membrane and eliminate high band that can bind your antibody and make competition with your specific signal.
-Use pico or femto ECL in order to enhance potential week bands you could have in the other sample.
This is the second Ab that I try, and I already set different exposure times.
I could easily try to cut out high bands to avoid non-specific binding of the Ab to polypeptides with higher molecular mass, but I really would like to understand if anyone has already encountered ~63 KDa extra-bands with commercial anti-GSTA2 antibodies, even from other suppliers.
More importantly, I would like to know if anybody has a clear idea of what such bands can contain (complexed GSTs, immature polypeptides, etc.).
Thank You so much for Your suggestions, Raphael.
Regards
Stefano
I havent worked with this specific antibody, however there are two things I can suggest.
(1) "If" the extra bands that you see are non specific bands then you can increase your NaCl Concenteration to 0.5M or 1M in the TBST (which normally has 152mM NaCl) I have done this before with other antibodies and the nonspecific bands virtually disappear.
(2) Second is if you carefully see your "nonspecific bands" they are in a difference of ~10Kd. I.e specific band at around 25, then supposedly non specif bands at ~35, 45,55,65Kd. You may want to look if your proteins are getting Ubiquitinated (Ubiquitin MW is ~8.5KD)
Very good suggestion! I think you got it. I didn't think about ubiquitination. Thanks so much.
Technical Service of Ab supplier offered some suggestions based on the data I provided.
Judging by the blot data and information about the protein, the Supplier suggested that the additional bands are non-specific. Given a quick search on its expression, GSTA2 is not expected to be highly expressed in the fibroblasts or neuroblasts. Moreover, the protein also is not reported to form multimers that would withstand the denaturing conditions of my gel. So non-specific bands would be the best explanation and the polyclonal rabbit antibody is prone to binding off target.
However, Deepak Kanojia's advise is always in my mind. May I confirm the hypothesis ubiquitination of GSTA2 by treating my fibroblasts or neuroblasts with the proteasome inhibitor MG132? In that case, would I see an increase in high molecular mass "non-specific" bands, reflecting an accumulation of poly-ubiquitinated GSTA2?
Thank You very much in advance.
Stefano
I would suggest, to make sure that these bands are not because of cross reactivity of antibody. If they are not, evaluation of ubiquitination following treatment of all three cell lines with proteasome inhibitor would be an ideal experiment. (I looked at GSTA2 protein and it appears that there is a "weak" possibility of ubiquitination at c-terminal)
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