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1. Could you confirm if reducing and denaturing conditions have been tried? We recommend to use antibodies in reducing and denaturing conditions in WB unless it recommends to use non denaturing and reducing conditions on the datasheet. This will ensure that the proteins are in the correct conformation to run at the correct molecular weight and be detected by the antibody. Samples were run in reducing and denaturing conditions using SDS and DTT.
2. Could you confirm what size are the 3 bands in the blot in the eluted samples? And what size bands were you expecting? The bands in lane 3 are presumably the eluted antibody:˜120 kDa, ˜50 kDa and ˜24kDa
3. How was the sample eluted from the IP column? Which buffer was used? Samples were eluted by boiling in denaturing SDS sample buffer.
4. Which lanes are the whole cell extract in, is this lane 1 and 2 (input A and B?). Yes. These lanes should give various signals, since I doubt that there is no methylated protein in the extract.
5, Has the transfer to the membrane and quality of the sample been assessed with a loading control in the whole cell extracts? Transfer to the membrane may be different from the eluted samples if they are in different buffers etc). Yes, there were more samples on the gel and these blotted and performed just fine with the respective antibodies in all cases. Blotting was also confirmed by Ponceau staining.
Asked on Dec 05 2012
Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.
I look forward to hearing from you with details of how you would like to proceed.
Answered on Dec 05 2012