Anti-GIP antibody [04] (ab30679)

Dear Abcam,

We have recently ordered your monoclonal GIP ab (ab30679) with the intention of using it for ELISA. We have tested the binding between the antibody and I-125 labeled human GIP(1-42) in free solution. Unfortunately, we are unable to detect any binding at all. Do you have any possible explanation for this? Furthermore, on the data sheet a few dilutions are recommended for ELISA. Would it be possible to recieve a copy of your ELISA protocol to see how the procedure works in your hands?

Looking forward to hearing from you. We are very interested in making this work.

Best regards,

ANSWER:

Thank you for your enquiry regarding our product ab30679. I am sorry you have had difficulty obtaining satisfactory results from your binding experiments with this antibody. Although this antibody has been tested by ELISA, it has not been tested for this type of binding experiment, so I cannot guarantee it will work. However, I would like to make a couple of suggestions that I hope may help. Unfortunately, there are no details of the specific epitope this antibody recognises, but it could be that your I-125 label is within the antibody binding region of the protein, and therefore interfering with binding of the antibody. You may also like to check the pH of the solution you are binding in. Most antibodies bind more efficiently at pH7 to pH8.

If you have problems performing the ELISA, please do get back to us and we will be very happy to offer technical support. You will need to fill in the following technical form and send it back to us:

http://www.abcam.com/index.html?section=elisa&pageconfig=technical&mode=questionaire

As requested, I have copied details of the laboratory ELISA testing procedure for this particular antibody below.

I hope this helps. Good luck with your experiments. Please don't hesitate to get back in touch with us if you need to.

ELISA PROCEDURE:

ELISA, binding to directly immobilized GIP:

Coat: GIP in 2 ug/mL

Apply antibody 2nd layer: Simply start a 2-fold serial dilution from 1.0 ug/mL

(Alternately apply ab30679 in concentrations of: 10 ng/mL - 20 ng/mL - 40 ng/mL - 150 ng/mL - 300 ng/mL - 400 ng/mL and 800 ng/mL)

Detect bound ab30679 with HRP conjugated polyclonal anti-mouse Ig antibody

Color development and stop solution applied according to local laboratory standard reagents)

Inhibition ELISA, binding free GIP in solution

Coat GIP in 2 ug/mL

Inhibition of 0.1 ug/mL ab30679 to the GIP coat with free GIP in concentrations from 10 ng/mL - 2 ug/mL

Detect bound antibody with HRP conjugated polyclonal anti-mouse Ig antibody

Color development and stop solution applied according to local laboratory standard reagents.