Anti-TEM8/ATR antibody (ab21270)

Thank you for contacting us. We have not tested ab21270 for cross-reactivity with CMG2, but it would not be expected to react due to 23% sequence homology with the immunogen. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your reply and providing more information and a whole blot image. Your problem puzzled me greatly and I also asked other colleagues to help understand what might be happening. Here are a few of our thoughts: -For the western blots: you said "The band for TEM8 is seen as a doublet because it is a re-probing after using the monoclonal. When you strip blots, you take off the secondary antibody but not the primary, so you see it as an “empty space”." Have you tried using the antibody on a fresh membrane? Stripping can damage the proteins and therefore prevent them from being detected by the second primary antibody. Furthermore if there is still primary antibody from the first experiment the second primary antibody will have more problems binding to the protein. -The Abcam antibody has not been tested on human samples, and as the protein is expressed in different isoforms/ splice variants we are wondering if the Abcam antibody is better at detecting one splice variant over an other, hence in our samples of mouse kidney it can detect the protein well in the three isoforms while in your samples the expression pattern may be very different. We would recommend to run a positive control of mouse kidney whole cell lysate to determine if this is the case. For the Immunoprecipitations: -The small strong large band in the IPed samples (HA and Poly IPed) and reprobed with the polyclonals could be the 60kDa form of the protein, and the strong large band could be the 45kDa form of the protein. As the poly has not been tested in IP, it's very hard to know if it is working optimally for this application. Again, having a fresh membrane would be useful to see exactly what bands are due to what antibody. As the antibody has not been tested in human samples I think the best recommendation I can make is to run a positive control along your samples; we do not guarantee ab19387 to work in human but I would be more than happy to offer you a refund or another antibody if you still have a problem with the positive control.

I'm sorry to hear you are experiencing unsatisfactory results with ab19387. I would be happy to consider offering you a replacement vial of ab21270, however before I can do that I would like to investigate this matter further to make sure that the problem with ab19387 could not be solved with a few protocol modifications. The quality of our products is of utmost importance to us and should we find out that ab19387 is of poor quality we will not hesitate to remove it from the catalogue. Can you please click on the link below which will take you to a short protocol questionnaire to help you put the information together, and could you please put it to my attention so I can personally look into this matter as I already have in hand the images you kindly provided. https://www.abcam.com/index.html?section=ip&pageconfig=technical&intAbID=19387&mode=questionaire I am already trying to understand what might be happening and would be very grateful if you can clarify a few points: -there are several bands on all blots, I'd be very interrested to know their sizes but with only one MW marker am finding it hard to determine this (are the bands above 78kda close to 100kDa for example, or 80kDa?) -Looking at the western blots of the lysates, I see one band with the Mono (I guess this is the 80Kda band), but two bands both with the poly and the HA tag antibodies. I am wondering if the HA tag is not also a poly. In general polys do pick up better various forms of the proteins and it could be that those two polys are picking up cleaved forms of TEM8. Could this be the case? -Looking at the IPed reprobes, it seems again to me that the poly anti TEM8 and the HA tags are giving similar band patterns, wether it was an IP with HA antibody or the poly. So with the IP with the poly it seems to me that the reprobe with the HA does give something, and as the TEM8 is expressed not endogenously in your cells I am wondering if again it could be because of a cleavage of the protein. Interrestingly IPing with the mono did not give a band at 80kda, so I conclude that the mono does not work well in IP, is this your opinion too? I would be very grateful if you can let me know your thoughts on those points so I can better understand what might be happening in your experiments.

Thank you for your enquiry. Based on prediction software, the epitope for the anti-TEM8 is extracellular. I hope this information helps. Please do not hesitate to contact us again if you need anything further.

Thank you for your enquiry. The peptide was not selected from the transmembrane domain of TEM8. Unfortunately the exact sequence of the immunogen is proprietary. Please let me know if you have any more questions.

Thank you for your enquiry. I am looking into the details of the immunogen for you. Meanwhile, I can tell you that this antibody recognizes all three isoforms of the TEM8 protein. As soon as I have any further information for you regarding the immunogen, I will let you know. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.