Anti-cIAP2 antibody [E40] (ab32059)

Thank you

Yes Ill be working tomorrow. Please ship.

Id like to run one wb before going on the holidays.

Merry Christmas and Best wishes for a Happy New Year!

ANSWER:

Ok great, this will ship out tonight. Please keep me updated, and I look forward to hearing from you!

Hello again, Well actually two of my coworkers in the lab agreed to run separate experiments to make sure that there is nothing wrong with the protocol. Antibody that you are offering would definitely be useful. We can try and as a matter of a fact I have some nuclear extracts that I can run with that antibody. Thank you

ANSWER:

Thanks for getting back to me. I'm sending a vial of ab23423 free of charge on the order ***, which we have in stock and can be delivered tomorrow. Will your receiving department be open tomorrow, or should I set it for a later delivery? Please keep me updated about your coworkers' results using the older antibodies and your results with this new one. I am hopeful that one of these will work! Happy holidays and best wishes.

Hello,

In my first email I mentioned that left picture with high background is the membrane incubated with monoclonal antibody. The right picture represents the polyclonal antibody.

I did increase the contrast so the band would become visible.that is the reason having high background.

Thank you

ANSWER:

Thanks for your reply and for your patience while i have been in touch with the lab. I have heard back from the lab, and I don't see anything unusual in their protocol so I don't have a great explanation for these results. At this point, I would suggest trying a different BIRC3 antibody (though I know you've already tried 2). I've looked through the antibodies that we have to this target, and I think that ab23423 looks promising. The WB image on the datasheet shows some extra bands (thought to be cleavage fragments or non-specific bands), but we only have 1 complaint on this antibody since it has been sold. Ab23423 has also been used in 3 references. As I mentioned before, these antibodies are covered by our guarantee, so I can issued a credit or refund if you would prefer. I apologize for all of the difficulty with these antibodies, and I'm hopeful that we can identify one that will work for you. I look forward to hearing from you. Please let me know how you would like to proceed.

Hello again, I did load 10 micro liters of Daudi cell lysate. As far as I understand it does correspond to 20 micro grams of the protein load. There is a band using BIRC3 monoclonal antibody which I think corresponds to the protein, but there were no bands on HELA and A431 cells lysates. On the other hand there were very faint bands using polyclonal but unlike the monoclonal antibody, polyclonal did show some bands in the HELA, A431 as well as Daudi cell lysates.Although Daudi cell lysates showed several bands. Two of those were too close to the real band. I dont really know what is the reason. I used 5% BSA to block the membrane and then I diluted my antibodies in the PBS-Tween 0,1%. I incubated overnight. ECL kit is new. I used it second time during this experiment. I also use fluorescence antibodies and tried those but unfortunately didnt get anything either. I still have those membranes and can try to run the GAPDH to make sure that the ECL is fine. ALthough people in our lab used that kit afterwards and didnt have any problems. Refund sounds great, but I would rather get any signal since this is a very critical experiment in my project. I need to get any data and if you can help me fix this issue that would be fantastic. Thank You  

ANSWER:

Thank you for your reply and for sending the additional information.

I am in touch with the testing laboratory to ask for further suggestions to get the correct results. I do have a few more questions, just to make sure I understand the images that were sent. Could you possibly label the molecular weights of the ladder? And also to clarify, the left membrane with the high background is the monoclonal or the polyclonal? I apologize for the inconvenience but I have conflicting information in my notes and I want to make sure the details are accurate.

I will get back to you once I hear from the lab, and please let me know if you have any questions. I look forward to hearing from you and resolving this promptly.

Hello again, Im sorry that you couldnt open the gel file. I converted it into bmp file and added some contrast so the bands would become visible. This is all after 20 minutes of exposure with ECL. Thank You  

ANSWER:

Thanks for sending the image again. I'm sorry to see that the results are so poor with this replacement antibody. Do any of the bands on the blot correspond to the expected band size of cIAP2? How much lysate do you load into each well? The testing laboratory uses 5% non-fat dry milk instead of BSA to block the nitrocellulose membrane, and some antibodies are sensitive to blocking agent so it might be worth a try. Do you also dilute the primary and secondary antibodies in solution containing milk or BSA? This can be removed if so. Have you tried blotting for a loading control protein like beta actin on the same membrane, to check that the HRP and ECL kit are working well? We do guarantee this replacement antibody to work as well, so if the results do not improve I would be happy to issue a credit or refund for your original purchase. Please let me know if you have any questions or if there is anything else that we can do for you.