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BATCH NUMBER 81755 DESCRIPTION OF THE PROBLEM While looking for arginine methylation of SR proteins, I unexpectedly saw an ab412 western blot signal for the large subunit of RNAP IIO. Using chromatographically purified preparations of RNAPIIO and IIA from Hela cells, I surprisingly saw a signal only with the IIO isoform and nothing with the IIA isoform even though they are essentially identical except for post-translational modifications - namely extensive serine phosphorylation of the the C-terminal repeat domain (CTD). I thought that perhaps this was due to arginine methylation of the Pol II large subunit. However, when I tested a GST-CTD fusion protein expressed and purifed from bacteria, which is not modified, nor detected at all by ab412 with 200-400 ng GST-CTD protein, and subsequently phosphorylated the bacterially expressed GST-CTD with a highly purified CTD kinase (P-TEFb), I once again saw an ab412 western blot signal specifically with phosphorylated species which migrate much more slowly on SDS-PAGE gels. This result, specifically, and additional experiments involving phosphatase treatments and an IP all lead me to the conclusion that ab412 is cross-reacting in a very specific fashion with a phospho-serine epitope on the CTD of RNAP II. Granted, the amount of protein I am using is often fairly high, 100 ng to 200 ng per band, although I think as little as 5-10 ng is barely detectable. Also, I am using a 1:100 dilution of ab412 for most experiments although 1:500 gave a detectable signal by ECL. I am curious if you know whether this has ever been reported before. Certainly, this does not sound like good news but due to the nature of RNAPIIO association with chromatin it may be important. It also doesn't make much sense to me. Is it possible that there is contamination with PolII phospho-CTD antibody? The sensitivity for phospho CTD I am seeing with ab412 is not nearly as high as phospho-CTD antibodies H5 and H14, although the specificity is quite remarkable. I would appreciate any comments you have pertaining to my conundrum. SAMPLE bacterial expressed/purified GST-CTD (RNAP II C-terminal domain) Hela cell preparations of highly purified RNAP IIO and IIA PRIMARY ANTIBODY ab412 clone 7E6 mosuse monoclonal IgG1 ChIP grade- anti mono/dimethyl arginine - ascites SECONDARY ANTIBODY anti mouse IgG made in goat - 1:2000 DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED I havnt tested a true positive control so I don't know if the signal for pol II CTD is greater or less than normal, but my 1:100 dilution is being used by others. ANTIBODY STORAGE CONDITIONS 4 C SAMPLE PREPARATION proteins are all relatively pure and stable. Normal conditions. AMOUNT OF PROTEIN LOADED 5-200 ng ELECTROPHORESIS/GEL CONDITIONS reducing 8% SDS PAGE TRANSFER AND BLOCKING CONDITIONS 5% milk for 30 min. Not likely to be a problem as I see antibody specificity. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6+ HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
Asked on Feb 22 2005
After careful investigation of your feedback regarding ab412 we can confirm that the non specific binding is very unlikely and that the likely explanation for the signal for the large subunit of RNAPIIO is overloading of the well with the protein. Furthermore, the GST tag on the fusion protein may mask the recognition site and the purification process may have altered the protein structure. To experimentally confirm this a positive control would be best, we would recommend estradiol treated cells (human HeLa or mouse MCF7 cells) and to run lysates of those along with the purified proteins. We would like to also recommend using BSA (5% in TBST, 1hr) to block rather than milk as this is recommended for experiments with kinases. We are happy to say that ab412 is very specific and recognises only the asymmetrical di methyl arginine (not symmetrical). We hope this information helps, please do not hesitate to contact us again if you need further assistance,
Answered on Feb 24 2005