Anti-Rad21 antibody - ChIP Grade (ab992)

Dear Abcam,

We have been struggling western blot for MeCP2 with the one below.

Although we see bands in our blot usingab2828 below. Bands seem to be non-specific. Could you help with us?

Thanks.

ANSWER:

Thank you for your enquiry regarding a2828 and ab992 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with these two antibodies.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

a2828: Anti-MeCP2 antibody - ChIP Grade



This item was purchased almost 2 years ago (Order No: 70326, 10 Jun 2010) and I regret to inform you that the Abpromise for this antibody has expired.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) MeCP2 is localised to the nuclei so try to prepare nuclear lysate instead of whole cell lysate.

2) It is worth testing different dilutions as well (1/700 or 1/500) to see if the intensity of the signal is getting stronger or not

3) Not sure what cell line was used, this information seems to be missing in the e-mail. It would be worth running cell lysate from HeLa, AtT20 or U2OS.

ab992: Anti-Rad21 antibody - ChIP Grade



The protocol looks fine to me and since this antibody was purchased on 31Oct 2011 our Abpromise will certainly applies. Here are a few comments and tips I could make which may help improve the signal.

1) Try to increase the final concentration of this primary antibody on the datasheet, it is recommend using 2 µg/ml.

2) Rad21 is localised to the nuclei so try to prepare nuclear lysate instead of whole cell lysate.

I hope this will be useful for you. Should you still have any problem with this ab992 after following these suggestions, then please do not hesitate to contact our Technical Department again our Abpromise will apply for ab992.

Hi,

Could you please tell me what alternatives you have?
We selected this antibody because it was a mouse monoclonal and which would allow us to do Co-staining and Co-IP with other rabbit antibodies we have.
Do you have any other mouse antibodies for human RAD21? If not, do you have any other good antibodies against hRAD21?

Thanks

ANSWER:

Thank you for your response.

Currently, we only have rabbit polyclonal antibodies (5) against Rad21, and no mouse monoclonal available.

ab42478 and ab992 have been tested in ICC/IF and IP applications.

I hope this helps and if I can assist further, please do not hesitate to contact me.

don't these two statements from the datasheet contradict each other:

1. This antibody detects a band at 130kDa corresponding to Rad 21 and also a band of unknown origin at 75kDa.

2. WB: 1/500 - 1/5000. Predicted molecular weight: 72 kDa.

unless 75kda is the unprocessed precursor and the protein is phosphorylated?

thank you.

ANSWER:

Thank you for your enquiry. I have been in contact with the originator of this product, and the band at 130 kDa is the Rad 21 band, and the band at 75 is unknown. I do not know exactly why Rad21 runs a larger band than is predicted.

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

Customer Question: I was wondering what synthetic peptide was used to raise the antibody. Based on sequence homology, will it cross react with Scc1/Mcd1 in S. cerevisiae, a Rad21 homolgy?

ANSWER:

Thank you for your enquiry. This antibody is very unlikely to react with S. cerevisiae. The synthetic peptide sequence for this antibody is proprietary. I cannot give you any more information than that which is already on the datasheet. Please contact us again if you require further assistance.

Hi Charlotte,

Could you tell me more detail about the epitope location?

Thanks,

ANSWER:

Thank you for your enquiry. This Ab is a polyclonal Ab, as such there will be a number of different IGs present that may all recognise different epitopes contained within the immunogen.