Anti-bacterial Alkaline Phosphatase antibody (HRP) (ab7319)

I do incubate at 4C overnight. The higher concentration of antibody produced more background rather than specific binding, even when given higher BSA concentration in the blocking buffer.

Could I try the monoclonal anti-bacterial alkaline phosphatase antibody [BAP-77] ab49793 as a replacement? The suggested use for this antibody says a 1:20,000 dilution for western works, so it may be more effective against denatured protein.

Do you know for sure if either antibody was prepared against denatured protein, out of curiosity?

ANSWER:

Thank you for providing that extra information.

As requested, I am sending ab49793 as a free of charge replacement. Your new order number is 1080758 and you should have it by 10.30am tomorrow morning. Unfortunately all of the information I have regarding the immnuogen is that a fusion protein was used, I do not know anything else.

If there is anything else I can help you with, please let me know.

Inquiry: Hi, I am using your Anti-bacterial Alkaline Phosphatase antibody (HRP) in both ELISA and Westerns. I have a strong positive result for my protein in the ELISA from both whole cell culture, as well as supernatant. In western blots however, I am not seeing a band in the supernatant fraction. Is this antibody more active against natively folded protein, so it won't work as well in the western blot? I have used both 2% and 5% BSA block for 2 hours, and serial dilutions of antibody from 1:3000 to 1:200,000 to test which is most effective. Washes were done with .1%TBST, 6 times for 10 minutes each. Substrate is Supersignal west Femto max sensitivity from Pierce. Any advice on this would be appreciated. Thanks!

ANSWER:

Thank you for contacting Abcam.

I am sorry that you are experiencing some issues with ab7319 in western blot.

I was wondering how long you incubate your samples for, do you do overnight at 4C? Also we have some notes that suggest that the optimal concentration range to use this antibody at is between 1/500 - 1/2000. This may indeed be because it is less efficient at recognizing the denatured form of the protein.

The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

Looking forward to your reply and helping you resolve this issue.