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Customer inquring about IHC-P on human tissue for detecting BMPR2. Also for detecting TRPM8 via ICC and western blot. Customer asked for protocol tips and antibody suggestions.
Asked on Nov 28 2012
Thank you for contacting Abcam.
We spoke earlier about two antibodies:
anti-BMPR2 ab78422 (https://www.abcam.com/bmpr2-antibody-mm0060-9a10-ab78422.html)
anti-TRPM8 ab109308 (https://www.abcam.com/trpm8-antibody-epr41962-ab109308.html)
You mentioned you were interested in testing ab78422 in mouse via IHC-P prior to using the antibody in human samples. Since this would be a mouse on mouse stain, there will be a high level of background due to the mouse antibody binding to native IgG's present in the mouse tissue. Also, our AbTrial program does not apply to mouse on mouse applications. Thus, I highly recommend testing the antibody in your human sections, perhaps sections that are not as crucial to your experiment.
Additionally, purchase of ab78422 would entitle you to a free rabbit monoclonal antibody through our RabMab promotion. Details of this promotion are given below:
Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447
Thus, you would be able to receive ab109308, the anti-TRPM8 RabMab that we looked at together earlier with the purchase of ab78422.
We also have anti-mouse and anti-rabbit secondary antibodies that could be used for ab78422 and ab109308, respectively.
We also discussed some IHC-P protocol information. I've attached our protocol book which includes IHC-P protocol information as well as other applications. Specifically for ab78422, we discussed attempting a proteinase K enzymatic antigen retrieval. Information regarding a proteinase K antigen retrieval is given below. I also recommend primary antibody dilutions of 1/50, 1/100, and 1/200 to see which dilution gives the best signal to background ratio.
Proteinase K antigen retrieval information: http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm
We also discussed the western blot bands that you were seeing for TRPM8, with 2 bands appearing (1 at the expected 130 kDa and 1 at 60 kDa). Since the TRPM8 protein is a homotetramer, the 60 kDa band is most likely a homodimer composed of half of the TRPM8 protein. In order to reduce the likelihood of this lower weight band occurring, you can also attempt denaturation in SDS at 70oC for 5-10 minutes rather than boiling at 95-100oC.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Nov 28 2012