Anti-MBD1 antibody - ChIP Grade (ab3753)

We purchased MBD1 antibody from Abcam (see below) and tested this antibody. Attached is the film. I found at least two bands in the tissues from spinal cord (SP) and cortex (CO) in WT mice . Moreover, same bands are also found in MBD1 KO mice.

It appears that this antibody is non-specific. Please let us know what Abcam thinks about.

ANSWER:

Thank you for contacting us and bringing this to our attention.

I agree that the antibody you received appears to be non-specific, according to your data with the full-gene knockout. We have not received other reports of western blot non-specificity for this antibody. I will be happy to replace it. I suggest ab124519.

Has followed suggestions to reduce background in ICC, now there is no signal at all.

ANSWER:

Thank you for your call today and for letting me know about the results with these antibodies after trying the alterations that we previously discussed.

Please let me know about the results after trying the most recent protocol alterations that we discussed, such as removing the goat serum from the secondary antibody diluent, and increasing the concentration of primary antibody. I hope they will be useful, but if not then I will be happy to help you further.

Have a wonderful evening and let me know if there is anything else you may need.

High background using antibody to stain MCF7 cells.

ANSWER:

Thank you for your call today and for letting us know about the trouble with ab3753 and ab115692.

As we discussed, I would recommend further diluting the antibody and blocking with 10% serum to reduce the background with these antibodies. Please keep me updated about the results after trying these suggestions, and I will be happy to help you further if necessary.

Please let me know if you have any questions or if there is anything else that I can do for you. Have a great day!

I attached the report.

He has to graduate this year using ab3752.(Actually it remains only 2 weeks).

So please check this results as soon as possible.

He tried many time to get good results using ab3752, so if possible, he wants to obtain other antibody not ab3752 .

I wait your reply.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that your customer has been having difficulties with this antibody. The result that your customer has been obtaining are most interesting. I have read your customer's technical questionaire and I have a few comments.

They mention that they are applying the antibodies at a dilution of 1:100. We actually recommend that the antibodies are applied at the following dilution; ab3752 (MeCP2) at 1000, ab3753 (MBD1) at 1:1000 and ab3754 (MBD2) at 1ugml-1 (1:1000). These are considerably more dilute than your customer is applying and therefore I would like to recommend that they try applying ab3752 at a dilution of 1:1000. This often serves to improve the specificity of the antibody.

I would also like to recommend that your customer tries a no-primary control experiment. There are extraneous bands of a similar size in all three blots. This background may be attributable to non-specific activity of the secondary antibody.

Should a dilution to the antibody and verification of the secondary antibody not improve the results that your customer has been obtaining please get back in touch with me and I will arrange for a replacement vial or credit note to be raised (provided that the antibody was purchased within the past 120 days).

One of our customer bought this product and wants to use it for ChIP. But there is no proper dilution factor on the datasheet. So he would like to get some information to do ChIP. Because he has to use a lot of this antibody for his experiment, he wants to reduce the loss of antibody.

Please let me know proper dilution factor or information for it.

ANSWER:

Thank you for your enquiry.

This antibody was upgraded to ChIP grade and suitable for chromatin immunoprecipitation following the two publications below. It has not been tested in house.

Ballestar E et al. Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. EMBO J 22:6335-45 (2003). PubMed: 14633992

Fournier C et al. Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J 21:6560-70 (2002). PubMed: 12456662

In actual fact if you consult Ballestar E et al. it is clear that they employed a protocol developed in Gregory et al. 2001. From consultation of this paper it is apparent that to 125 to 250 ug of precleared chromatin they added ~5 ug of affinity-purified antiserum. Given that this is whole serum I would recommend that a titration of the antibody is performed within an initial range of ~2-10ul and adjust your future experiments accordingly.

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