Question (68138) | Anti-CD81 antibody [EPR4244] (ab109201)

Go to datasheet (ab109201)

Question

Good evening,

I am contacting as I took advantage of your rabbit mAb offer by ordering the anti-CD81 (ab#109201). I tested it by immunofluorescence as well as western blot and unfortunately I couldn't detect CD81 by WB and it wouldn't give me the usual staining by IF in HeLa.
I thus decided to run it against the usual anti-CD81 that I am using by IF in CEMss T cells (CEM cells that are prone to form syncitia) and I couldn't observe colocalization.

Please find enclosed a PPT in which I included images that I took last week.

Let me know what you think about it.

Best regards,

Answer

Thank you for contacting Abcam.
I am sorry to hear that this antibody is not working for you. As this was a free antibody given through our RabMab promotion, we cannot issue a refund in this case. However, if we cannot work with you to get ab109201 to work as indicated on the datasheet we will happily send you a free of charge replacement of the same antibody or another of your choosing.
Thank you kindly for including the ppt file with your IF results. My main concern is with the lack of a band in the WB, since there is definitely a positive signal in your IF, just not colocalized with the other CD81 antibody you used. Since these two antibodies were raised against different immunogenic sequences for the same CD81 protein, it may be that some cells express CD81 in a form more recognizable by one antibody over the other.
Thus, in order to help troubleshoot these problems, could you provide details regarding your sample, protocol and troubleshooting steps that have been taken, specifically for WB. These will not only help me to fully understand the problem but are essential should further testing of this product be needed as well. Could you also provide the Abcam order confirmation number or the PO and date of purchase for this product?
Based on the information you have provided, I have a few recommendations. According to the Human Protein Atlas database for CD81 (http://www.proteinatlas.org/ENSG00000110651/cell), HeLa cells have very weak, if any, expression of CD81. The positive control cell lines that we have tested are Ramos, JAR, U87 MG, IM 9, Peripheral blood mononuclear cell (PBMC), C6, PC 12, and RAW264.7 cell lysates. I highly recommend trying one of these positive controls. I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.
Below is a link with further troubleshooting steps if you have not performed them already:
https://www.abcam.com/index.html?pageconfig=resource&rid=11352
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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

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