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For your knowledge, I enclose the comments of two reviewers to a paper that we submitted to an international journal. We addressed the role of TRPM7 in endothelial cells and used your anti-TRPM7 antibody Ab729 e 72155 . Our manuscript was rejected because the antibodies recognize a band with a MW around 160 kDa and, although this band disappears after silencing with specific siRNA, this jeopardize the whole work.
I would appreciate your comments.
Reviewer 1. The authors use the anti-TRPM7 antibody from Abcam for their experiments. The western blots shown in nearly all the figures identify TRPM7 as a 150-160 kDa band. The molecular weight of the full length TRPM7 protein is roughly 220 KDa. It is possible that the 150 kDa fragment shown in the manuscript is a proteolytic fragment of TRPM7. In support of this view is the fact that knockdown of TRPM7 reduces levels of this band in Figure 4A at 24 hours.
While it is possible that the 150 kDa fragment is from TRPM7, the possibility exists that the 150 kDa band is not in fact TRPM7. The authors need to validate the Abcam antibody by showing that heterologously expressed TRPM7 is recognized by the Abcam antibody. In addition, the authors should show that the 150 kDa band in endothelial cells is recognized by a second TRPM7 that recognizes second epitope (another region on TRPM7). Without this data, the data presented is not convincing”.
Reviewer 2. The antibody used in the study stain a protein band of about 160 kDa, whereas the molecular weight of TRPM7 is 212 kDa. Thus, this is not convincing that western blots actually monitor levels of TRPM7. This is a critical issue because the study relies heavily on Western blot analysis of TRPM7 protein expression.
Asked on Nov 07 2012
Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.
I have read the details you have kindly provided and would like to investigate the complaint further. Could you send us the publication or the data you have gained with this antibody? Could you also fill the attached questionnaire?
Thank you very much for your cooperation. I will look forward to hearing from you soon.
Answered on Nov 07 2012