Anti-M6PR (cation independent) antibody [2G11] (ab2733)

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Thank you, I received the data this time. For a protocol modification, I suggest trying preparing a sample by heating at 60C for 10 minutes instead of 95 for 5 minutes to minimize aggregation of the trans-membrane domains. It is strange that both antibodies give you this result, and I still think there may be some issue with the sample entering the gel and/or transferring effectively, even though the standards do.

Another modification is to remove methanol from the transfer buffer and add SDS to a final concentration of 0.1%, to encourage transfer of this large protein.

Are you confident that the secondary antibody is not reacting with the samples? Is the secondary specific for a subclass of mouse IgG?

These modifications are unlikley to remove the band at 70 kDa. We do have other antibodies against the receptor, so if these modifications fail, I can send a vial of something else to try, such as ab32815 or ab124767.

Thank you for bringing this to our attention. I will need a few more details of your protocol. I was not able to open the file you sent.

Can you please tell me how you prepared your lysates (did you use protease inhibitors), and how you heated the samples to denature. I am concerned that the receptor may have aggregated upon boiling and did not enter the gel effectively, or did not transfer to the blot well. Did you check for efficacy of transfer with a Ponceau Red stain or equivalent?

How much sample did you load per lane of the gel? How did you block the membrane? What concentration of antibody did you apply to the membrane and how long did you incubate with the antibody?

Thank you for contacting us regarding ab2733. I have confirmed with the laboratory that this particular antibody is only batch tested in IF, but has been tested in WB. I have attached an image for you. We have recently done some optimisation with TA gels (and 0.45 um membranes) and we found using 10% methanol +0.04% SDS or 10% ethanol +0.04% SDS for the transfer buffer worked really well. We also found that it was best not to heat the lysates to 100C for TA gels (no heating was best but 60C was ok). We haven’t tried this procedure with this ab though so I can’t say if it will help solve your problem for sure. The presence of SDS increases solubility of membrane proteins and high MW proteins allowing efficient elution from the gel. As standard we denature at 95C. Reduced denaturation temperature (60-70C) or no denaturation is often recommended for membrane proteins/high MW proteins to reduce precipitation and failure of the protein to migrate onto the gel during electrophoresis. I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions or if your results do not improve with this antibody.