Recombinant Anti-CD81 antibody [EPR4244] (ab109201)

Thank you for your reply and protocol information.
If we cannot get this antibody to work as stated on the datasheet we will replace it with a new vial of the same antibody or a different antibody of your choosing free of charge as indicated in our AbPromise guarantee. Given that, I have some more questions and protocol tips.
Are you reducing and denaturing your sample prior to Western blot? We recommend boiling for 5 minutes at 95oC in beta-mercaptoethanol or DTT + SDS prior to loading. Since there is a disulfide bond within the residues that are recognized by this antibody, if that disulfide bond is not broken via reduction/denaturation the antibody may not properly recognize its epitope.
For IF: In our experience testing our antibodies, Triton-X-100 is not appropriate for the use of membrane-associated antigens (such as CD81) since it destroys membranes. Since the specific epitope for ab109201 is extracellular, you should not have to permeabilize the cell membrane in order for the antibody to detect its antigen.
For IP: As stated on the datasheet, this antibody is unsuitable for flow cytometry, IHC-P, and IP, explaining why it did not work in IP for you. The unsuitability of this antibody in these applications where CD81 is in a native conformation is most likely due to the fact that the antibody was raised against a synthetic peptide corresponding to an extracellular domain of CD81 that contains a disulfide bond (forming a loop) that is harder for the antibody to recognize in native 3D form compared to a linearized (ie. reduced, denatured for WB) form.
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 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

Thank you for your participation in Abcam's post complaint survey.

Please let me know if there is anything else I can do for you.

Also, if you have further questions to any of our products (e.g. primary antibodies, lysates, secondary antibodies, kits, controls, gels, proteins/peptides, slides, biochemicals, conjugation kits), please let me know as well.

Vielen Dank für Ihre Antwort.

Es tut mir leid, dass Sie Probleme mit diesen beiden Antikörpern hatten. Wie per Emailbesprochen, habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer xxx fuer ab7090 und ab93485, und sollte diese Woche bei Ihnen ankommen.

Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.