Anti-Citrulline 抗体 (ab6464)

Thank you for contacting us. We currently have two antibodies that will detect the citrulline modification on any proteins: ab6464 and ab100932. While ab6464 has been validated in IHC on frozen tissue sections, we have not tested this antibody for use in WB. Similarly, ab100932 has been validated in WB but not in IHC. If you would like to test either of these antibodies, I am happy to offer you our 100% testing discount program. If you purchase the antibody for full price and submit an Abreview with your protocol and results using the antibody in an untested application, you can get a discount code for a free primary antibody on a future purchase. Whether or not the antibody works, the next antibody you buy from us would be free. If you are interested in this program, please reply and let me know which antibody you would like to test, and I will be happy to set up the code for you.

Thank you for your reply and this complement of information.

ab6464 has not been tested in Western Blot and may not be suitable for this application. This would explain why you don't see any staining with this antibody.
The information on the product datasheet as well as the encouraging positive results obtained in Western Blot suggest that ab100932 is indeed capable of targeting the citrulline independently of the amino acid sequence.

An optimisation of the incubation time and conditions for the antibody ab100932 in ELISA may be necessary in this case.

Please let me know about the new trials with the antibodies in ELISA. If the problem stays the same I will be happy to issue a credit note against the original order.

Thank you for contacting us.

The source of the antibody informed us that they have never tested the antibody by coating plates with fibrinogen.
Their testing are made using BSA and citrunilated BSA. However, the testing conditions are very similar to yours. The difference is that the lab do not perform a washing step before the saturation with BSA.
I attached to this email the results obtained during the validation of batch GR22466 of this antibody ab6464 as well as an ELISA protocol provided by the lab.

I hope this will be helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your email.

I am very happy to hear that the protocol optimizations improved the results and the antibody is now working fine.

I wish all the best for future experiments and research.

Thank you for your inquiry! We are well, thanks- Ihope you are well too!

The two products are from two different laboratories and therefore productions. They are both against bound citrulline and guaranteed by us in ICC/IF, ELISA, IHC-Fr for the ab6464 and ELISA, ICC, WB for the ab100932. If the customer wants to do Western blot, I can recommend the ab100932. If the customer wants to do frozen section immunohistochemistry, I recommend the ab6464.

Other differences are the information or the unit size. The ab100932 comes in 200ul and for the ab6464 we have the crossreactivity data against homocitrulline for example on the datasheet.

I hope this information is helpful. Please do not hesitate to contact us again should you have any other question.

Thank you very much for filling in the questionnaire.

The lab is using the belowprotocol to test the antibody. I noticed there are differences, particular concerning the buffers. The positive control is Citrulline-BSA.

Maybe this protocol will help improve the results. Please let me know how you get on.

ELISA protocol:

1 - Coating of Immunogen(BSA) (10µg/ml) in maxisorp well plates with a solution of sodium carbonate buffer 0.05M (pH 9,6), during sixteen hours at 4°C.

2 -Saturation of well plates with of a solution of PBS (pH 7,35) containing 2.5g/l of BSA , 10% and 0.5% of Tween (one hour at 37°C).

3 - Wash with PBS containing 0.5% of Tween (PBS Tween) (three times).

4 -Antibody will be diluted (1/2,000 – 1/10,000) in PBS Tween containing 2.5g/l BSA, 200µl by well plate (incubating during 2 hours at 37°C).

5 - Wash with PBS Tween (three times).

6 - 200µl of peroxidase-labelled goat anti-rabbit diluted (1/10,000) in a solution of PBS Tween containing 1g/l of BSA, will be applied by well plate (during one hour at 37°C)

7 - Well plates will be rinsed with PBS Tween (three times).

8 - And finally the peroxidase will be developed by incubating 200µl by well plate of a citrate 0,1M/phosphate 0,2M (pH 5) solution containing 0.4% of OPD and 0.03% of hydrogen peroxide for ten minutes in the dark, after that, we will stop the reaction by the addition of 50µl of 2M HCl.

9 - The optical density will be measured at 492nm.

Competitive ELISA protocol:

The same protocol is used, but modifications occur in stage “4”. The day before the test, competition scales are made with each competitor (10-1 - 10-10g/l of BSA conjuguate) and diluted antibody. These tubes are incubated at 4°C overnight. The results are expressed as OD with competitor/ OD without competitor for each point of competition scale.