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The images that I send to you are T cells which express tones of CD81 that is why I didn't send any picture of HeLa cells (though based on our experience one can detect CD81 in HeLa cells).
The problem that I am having with the rabbit Ab is that he doesn't correspond to CD81 staining in T cells. CD81 is mostly express at the pm in those cells and I have been working to understand CD81 down regulation in HIV-1 infected cells.
IF staining protocol (standard condition):
Briefly, CEMss cells (T cells) have been allowed to stick on poly-L-lysine treated MatTek dishes for 2h then wash once with PBS, fix using PFA 4% (wash then) for 10 min at 37 deg C, permeabilized (PBS + 1% BSA + 0.2% Triton) for 20 min at 37 deg C, primary Ab were incubated 1h at RT (rabbit anti-CD81 diluted 100 times, while mouse anti-CD81 diluted 500 times), wash , incubated with secondary Ab at RT for 30 min, wash, 2 ml PBS. Then I analyzed my cells on a deconvolution microscope.
Western Blot (standard condition):
When I used the Ab for WB application, I used CEMss lysates (T cells line lysed in PBS-1% Brij 96/97 containing PI) (infected or not). Using your Ab I couldn't see anything but when reprobing using our anti-CD81 I would be able to see the bands … (Conditions: 10% SDS-acrylamide gel, migration 120 V (mini-Protean system), semi-dry transfer (25V for 1h30) onto PVDF membrane, wash once TBS-T, Block in 5% milk in TBS-T, primary ON at 4 deg C, was 3 times, secondary HRP for 30 min at RT, wash 3 times, ECL.
I also was doing IP of CD81 and I couldn't see anything either (I was using the rabbit Ab at a dilution of 1/1000). I was IPing using the ms anti-CD81 and try to detect with the Rabbit again nothing clear in my hands.
Let me know if you have more questions/concerns based on my protocol.
Thank you for your reply.
Asked on Nov 29 2012
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.
Answered on Nov 30 2012