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I have a control peptide that contains citrulline and the same one that has arginine. These peptides are conjugated to BSA and I have successfully used them by Western blotting (0.1Ug) to confirm that the Millipore kit that detects modified citrulline is indeed specific. This kit gave no background but is not available now so I need another source to detect citrullinated proteins. Not only does the Abcam antibody not detect a known citrulline, but it also gives a very high background on the membrane (PVDF). I originally used this Ab on blots containing fibroblast extracts using standard conditions. I tried changing conditions to increase the signal to noise ratio and eventually was able to see some bands when the Ab was at 1/4000, incubated in TBST containing 0.25M NaCl, 0.1%NP40 and 3% BSA overnight 4C. I also always see some negative bands (white) on the blots. These show up immediately, so are not resulting from the substrate being used up. However, the background, (the entire blot is dark) is so bad that the dark bands can only be seen if held up to the light and I can not scan it for quantification or publication purposes. The secondary used is HRP-conjugated anti-rabbit at 1/40,000 in 3% BSA/TBST. I use this secondary antibody for all my other rabbit primary antibodies and have never encountered problems with it. I detect with lumiglo-again no prior problems.
Asked on Dec 13 2012
I am sorry to hear that this antibody is not providing satisfactory results.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab100932. I would also appreciate if you can confirm some further details:
Typically a high background with white bands or "ghost bands" indicates a signal that is too strong. You can reduce the primary antibody even further and/or add additional washes after primary incubation. Another option is to incubate only 1 hr at room temperature with the primary. You may also want to switch to 5% milk for blocking, at times when the BSA is giving high background sometimes switching the blocking agent solves the problem.
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.
Answered on Dec 13 2012