Anti-Glucocorticoid Receptor alpha antibody - ChIP Grade (ab3580)

Phone call reporting problems using ab3580 in WB.

ANSWER:

Thank you for contacting us and reporting the problems you have been experiencing with anti-Glucocorticoid Receptor alpha antibody (ab3580). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As discussed over the phone, I have checked into how this antibody has been characterised by Western blotting. This antibody has not been characterised in-house by Western blotting but we have had reports of it being used for this application from journals published (please see the attached journal articles).


If you wouldn't mind, could you fill out thequestionnaire I have attached to this email so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results with ab3580. This information will also allow us to investigate this case internally and initiate any additional testing where necessary. If you could include an image of the results you have obtained that would be most useful.


If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. In order to do this we need the order number (or the delivery address and the approximate date the order was placed).

I look forward to receiving your reply.

Are any of your glucocorticoid receptor antibodies specific for the GR-alpha isoform? Is the predicted seequence of GR-beta short enough to be visibly different on a standard western blot?

ANSWER:

Thank you for your interests in our products.

Yes, we have a product (ab3580) that specifically detects GR-alpha from human, mouse and rat tissues and does not detect GR-beta isoform. I have attached a datasheet link to this product for your easy access.

As you know, the human GR exists in two forms, alpha and beta, which are thought to be the result of alternative splicing of a single gene. Sequence analysis indicates that the alpha and beta forms of human GR are 777 and 742 amino acids long, respectively. They are identical up to residue 727, after which they diverge. After ligand binding, the 94 kDa GR alpha isoform has been observed to translocate from the cytoplasm to the nucleus where it regulates gene expression. In contrast, the 90 kDa GR beta isoform does not appear to bind either glucocorticoid agonists or antagonists, and has been localized predominantly in the nucleus independent of hormone treatment in some human cell lines. Studies suggest that human GR beta might function as a dominant negative inhibitor of GR alpha activity.

I got a further question on the ChIP-grade GR antibody. You say that this antibody is guaranteed to work in ChIP. But I've had problem previously with the sonication and immunoprecipitation steps of the ChIP experiments. I've noticed that I need SDS (at least 0.5%) in the sonication buffer to get efficient sonication of my chromatin (using the bioruptor sonicator). This high detergent concentration sometimes interferes with the immunoprecipitation step, as some antibodies don't do well in such conditions. I've noticed that your published ChIP protocol does not include any SDS in the sonication buffer, which would result in incomplete fragmentation of my chromatin using the bioruptor. I am worried that adding SDS might affect the immunoprecipitation step. So, basically my question is in what ChIP conditions was this antibody tested? You say that this antibody (ab3580) was shown to be ChIP-grade by an external originator... but was protocol/conditions was he using? The published AbCam one or something else?

ANSWER:

Thank you for getting back in touch with me.

Yes, you are correct a high SDS content can potentially interfere with your antibody-antigen interaction by ChIP. However, our Abcam protocol has been optimized and developed by an experienced chromatin postdoc colleague in our lab. She also uses the bioruptor for the sonication step. With regards this antibody; I can tell you that Glucocorticoid Receptor alpha antibody - ChIP Grade (ab3580) was actually applied using ChIP in the following publication:

Ichijo T et al. The Smad6-histone deacetylase 3 complex silences the transcriptional activity of the glucocorticoid receptor: potential clinical implications. J Biol Chem 280:42067-77 (2005). PubMed: 16249187

I have emailed this publication in a follow up email for your information. You may wish to consult the chromatin IP methods employed.

I am interested in testing both ab3578 and ab3580 in a fish cell culture system, trout and sea bream hepatocyte cultures. There is no technical data available considering testing in these species. My question is economic. Is it possible to acquire smaller aliquot sizes in order to test the antibodies by western blot in these species. As no cross-reactivity will result in an expensive exercise >600E. Does this possibility exist at Abcam?

ANSWER:

Thank you for your enquiry. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement.

Should you decide to test an antibody in species or applications for which we do not have any information, please let us know how you get on and in return we will award you 50 points with the Abcam Loyalty Scheme which can be redeemed on a number of rewards.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Strong band between 66 - 98 kDa markers, therefore it is not the 94 kDa that would be present according to the datasheet.

SAMPLE HUVEC whole cell lysate

PRIMARY ANTIBODY ab-3580 at 4 microgrammes/mL in 5% milk/TBST

SECONDARY ANTIBODY Anti-rabbit HRP (Amersham) 1:5000 in 5% milk/TBST

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED We would like you to send us a positive control (either recombinant human GR alpha or HeLa cell lysates, which I believe are used as positive controls for GR alpha and COS-74 cell lysates, which would be a negative control since they are strongly positive for the beta isoform and not alpha) for this antibody since it is not performing as anticipated from the datasheet.

SAMPLE PREPARATION Boiled for 3 min in sample buffer (beta-mercapto, SDS, bromophenol blue etc.)

AMOUNT OF PROTEIN LOADED 20 microgrammes

ELECTROPHORESIS/GEL CONDITIONS 7.5 % Tris-Glycine reducing gel run for 1 h at

TRANSFER AND BLOCKING CONDITIONS Transferred at 100 V for 1.5 h (Trizma/Glycine transfer buffer)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? once HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ANSWER:

Thank you for the details that you have provided and I'm sorry to hear that you are experiencing difficulty with this antibody. As stated on the online datasheet, we do recommend using HeLa cell lysate as a positive control for ab3580. Without running a positive control, it is difficult to say what may be going on. However, I unfortunately can't send you the lysate free of charge.

If you have any additional questions, please let us know.