Anti-XPA antibody [5A2] (ab44)

I'm looking for some help on product info for your ab44 - Mouse monoclonal [5A2] to XPA. I will be attempting to perform imunoflourescence studies on some fibroblast cells. Some of the cells express XPA but it is truncated at aa - 204 (out of 273). I was hoping to find an antibody that would yield a NEGATIVE result in these cells. Your ab44 - Mouse monoclonal [5A2] to XPA literature does not mention the epitope or binding region. Do you believe that it will bind to a truncated 1-204 XPA protein? If so, do you have an antibody for XPA that binds to the C-term?

ANSWER:

Thank you for your enquiry regarding ab44. We have not mapped the epitope recognized by this antibody therefore we do not know if it will or will not recognized your truncated protein. All our other antibodies against XPA are likely to recognize the non truncated form (e.g. polyclonal antibodies) as well as the truncated form or we do not know where the epitope is.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

Any references on the sucsess of using ab44 (XPA ab) in immunofluorescence detection on cell cultures?

ANSWER:

Thank you for your enquiry. All the information that we have regarding this antibody is located on the online datasheet, and at this time, we are unaware of any publications/references that feature the use of ab44. If you have any more questions, please contact us again.

I have done the direct ELISA as directed by your protocol and I still do not see a signal. I have confirmed that the secondary antibody by itself gives a signal and also recognizes the primary XPA antibody. Do you have further suggestions?

ANSWER:

Thank you for getting back to us and sharing your recent results.

We are very sorry to hear that you are still having problem with this antibody. The protocol we have sent it to you is a general ELISA method.

Looking at our order record, we can inform you that we have sold several vials of this product without any problem. We get this product from an academic source and originally it has not been tested for ELISA. However, one of our customers reported in 2001 that it worked nicely on ELISA application. We published his comment on-line, please take a look at John Butler's review on the top panel of the datasheet.

We would advise perhaps to contact this researcher (you can send an-email to him directly by clicking on his name) to get some more details of his protocol and discuss this issue further.

Certainly, we can offer you a replacement vial - free of charge, it is possible that the vial you received may have gone off during shipping and/or storage. If you would like to get it, please do let us know.

We look forward to hearing from you soon.

ANTIBODY CODE ab44 DESCRIPTION OF THE PROBLEM No signal

SAMPLE human XPA 1?g

PRIMARY ANTIBODY Ab44 1:1000 dilution in 1xPBS, 5% milk 1hour at room temp Wash 3x with 1xPBS, 0.2% Tween

SECONDARY ANTIBODY Sheep anti mouse HRP or goat anti mouse HRP 1:1000 in 1xPBS, 5% milk Wash 3x with 1xPBS, 0.2% Tween

DETECTION METHOD 200?l o-phenylenediamine in 0.05M phosphate-citrate buffer with 0.03% sodium perborate

POSITIVE AND NEGATIVE CONTROLS USED Positive : Tag-1?mouse combination that uses the same secondary antibody. This works. Secondary antibodies are good.

I have used this ELISA procedure successfully many times for detecting Tag, DNA pola and RPA.

ANTIBODY STORAGE CONDITIONS Received cold. Aliquoted and stored in -20.

TYPE OF ELISA Direct ELISA

COATING WELL human XPA 1?g in water

BLOCKING CONDITIONS 1xPBS, 5% milk 10minutes at room temp Wash 3x with 1xPBS, 0.2% Tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have used two separate secondary antibodies.

ADDITIONAL NOTES This antibody was ordered through the Department of Biochemistry at the University of Iowa.

ANSWER:

Thank you for your enquiry. We are very sorry to hear that you are having problem with this product.

I have searched our database and found that this is a popular selling product and your feedback is the first we have received about it not working. Therefore, at this stage, I would suggest that there is either a problem with the vial you received, or modifications to your protocol are needed to obtain a positive result.

I would like to make the following suggestions:

It is very important to emphasize that when you coat the wells with the antigen, always use coating buffer instead of water.

For ELISA, we would suggest using 1% (v/w) BSA and the blocking should be at least for 60 min. 10 min is too short!!!

I will send you detailed protocol for you in my next e-mail.