Anti-GFP antibody (FITC) (ab6662)

I I have a question regarding one of your antibodies, the Anti-GFP (FITC-conjugated) (ab6662). I wonder whether it is conjugated with a secondary antibody fused to FITC or, if not, how the FITC is attached to the primary anti-GFP antibody. Is the FITC irreversibly attached to the primary antibody or can the FITC moiety attach to other primary antibodies used in the same staining protocol? Does the antibody solution contain free, unconjugated FITC molecules that can attach to other primary antibodies in the staining mix?

ANSWER:

ab6662 is directly conjugated to the FITC molecule, thus there is no secondary antibody present. The FITC molecule is attached directly to the Fc portion of the antibody via covalent bonds to amine groups. The FITC is not irreversibly attached to the antibody, however, because the bonds formed are covalent strong chemicals would be required which could also damage the antibody. Lastly, there is no unconjugated FITC in the vial; the only FITC present will be conjugated to the antibody.

Has this antibody been tested for cross-reactivity with other GFP variants? If yes, which ones?

ANSWER:

Thank you for your enquiry and patience. This GFP antibody will cross react with YFP, CFP, eGFP, but not RFP.

Please contact us again if you have any additional questions.

BATCH NUMBER 103393 ORDER NUMBER 46006612

DESCRIPTION OF THE PROBLEM No staining

SAMPLE Mouse mammary gland sections.

PRIMARY ANTIBODY Attempted multiple dilutions from 1:5000 to 1:50 with the GFP-FITC conjugated. Incubation times included from 1 hour to over night.

SECONDARY ANTIBODY none

DETECTION METHOD ImmunoFluorescense.

POSITIVE AND NEGATIVE CONTROLS USED The slides used were previous tested and confirmed with an anti-GFP polyclonal rabbit antibody.

ANTIBODY STORAGE CONDITIONS We received the antibody and immediately stored at 4C.

FIXATION OF SAMPLE 10% Formalin to fix, then paraffin embedded.

ANTIGEN RETRIEVAL Heat mediated with citric acid buffer.

PERMEABILIZATION STEP none

BLOCKING CONDITIONS 10% goat serum for 1 hour.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? none HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Dilution of antibody and incubation periods.

ANSWER:

I'm sorry to hear that you are having problems with ab6662, we have not received any other complaints about this antibody and I think the problem you are experiencing might be due to the lack of permeabilization or the antigen retrieval method.

I would like to suggest incubating the blocking agent and primary antibody in PBST (PBS with 0.3% triton x100). one of the users of ab6662 has put a review of the antibody on the datasheet and has used it in paraffin sections, he may be able to advise you on the best method of antigen retrieval and I would suggest also trying out an enzymatic antigen retrieval method.

If you still have problems please do not hesitate to let me know and I can arrange for a replacement vial to be sent to you or a credit note or refund if you have purchased the antibody in the last 90 days,

I need technical support for the product - ab6662 I recently purchased. In the datasheet, it is writen that this antibody was purified through an affinity column. May I know the elution condition you used? I plan to make a small antibody conjugated column to purify a GFP fused recombinant protein. Knowing your elution condition will help me determine the most gentle elution condition. Thank you.

ANSWER:

Sorry, we would like to keep this information confidential.

Does this ab work on cells fixed with methanol and/or acetone?

ANSWER:

To our knowledge, this antibody has yet to be tested in this application. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on and in return we will forward a reward of your choice, typically an Amazon gift voucher.