Anti-Anterior Gradient 2 antibody (ab22208)

TSPY has been tested two times by student in this lab using the same condition, same cell lysate as worked out by AGR2 from Dr. Charles. Ab doesn't work could be due to a couple of factors, original quality, shipping, etc. Based on my 20 years' working experience on WB, IH, to be frank, your original quality of the batch I ordered is highly suspected.

Regards,

ANSWER:

Thank you for your rapid reply. I understand that you are concerned about the quality of ab30927 and take your comments very seriously. Please be assured that should we suspect that the quality of one of our products is not of the highest standards we will not hesitate to remove that product from our catalogue. We have not received any other complaints about ab30927.

I have looked at the protocol you kindly provided for the testing conditions of ab22208 and, like Tom's recommendations for ab22208, would recommend to incubate the antibody overnight at 4C to maximise binding of the antibody to the protein. Your student should also try a 1/500 dilution, as the 1/1000 recommendation is a guideline and depending on the levels of your protein in your lysates you may need more antibody. I would recommend to block the membrane in 5%BSA and incubate the primary and secondary antibodies in TBST only. We find that some antibodies "stick" after too much blocking, and give non specific bands and do not bind to the correct band.

If you still experience a problem with ab30927 with the changes I recommend above, please do not hesitate to contact me and I can offer you a refund on your purchase of this antibody,

BATCH NUMBER 180821 ORDER NUMBER 152075

DESCRIPTION OF THE PROBLEM No specific band around 18Kd size but with more than 100Kd weak band.

SAMPLE Prostate cancer C81 cell stably transfected with Ebp1 of our interest. Crude cell lysate for WB.

PRIMARY ANTIBODY AGR2, 1:100 in 2% milk, incubated in RT for 2 h,

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS After aliquot, half store at +4 and half at-20.

SAMPLE PREPARATION NETN lysis buffer with P.I; heated before loading to gel. All the regular routine procedure.

AMOUNT OF PROTEIN LOADED Strong control Actin signal.

SECONDARY ANTIBODY Anti-rabbit, 1:10,000 in 2% milk RT 1 H

ANSWER:

I'm sorry to hear you are having a problem with ab22208. As noted on the datasheet, this antibody has not been tested for Western blotting, only ELISA and IHC. However, the following protocol modifications may improve your results:

1. Fully denature your samples by boiling in loading buffer for at least 5m.

2. Load as much protein as possible. The strong actin signal is a consequence of the relatively large amount of actin in your samples and the efficacy of your actin antibody.

3. Incubate the AGR2 primary overnight at 4C, 1/100.

4. Run a "no primary" control to determine if your secondary antibody is responsible for the band at 100kD.

Please let me know if this helps and do not hesitate to contact us for further advice.