Anti-beta Actin antibody [mAbcam 8226] (HRP) - Loading Control (ab20272)

We recently ordered two tubes of monoclonal beta-actin hrp antibody (#AB20272), and while we have had great success with this antibody in the past, our western blots with this lot had such high background that we could not detect any meaningful signal. Could we exchange these tubes for fresh antibody?

ANSWER:

Thank you for contacting Abcam.


I am sorry that you have been experiencing difficulties with this antibody in WB. I have checked our current stock, and this is the only lot currently available. I wanted to get some additional information regarding the protocol you have tested so that we may investigate further.


Would you please provide a summary of your protocol and sample preparation details? Also, did you test both vials? I look forward to your reply so that I may assist you further.

I would like to purchase Anti-beta Actin antibody [mAbcam 8226] (HRP) - Loading Control (ab20272) but I have a query about it first.

Can this antibody detect if the sample has been prepared using SDS or will it destroy the epitope?   Looking forward to hearing from you.  

ANSWER:

Thank you for your inquiry.

Unfortunately, we do not currently have information about use of this product with an SDS lysis buffer. However we do not believe that this type of treatment will have detrimental effects for the loading control.

I hope that you find this information helpful. Please let contact us if you have any further questions

In respons to your question, I have not heat the samples prior to electrophoresis. You think that heating will improve the detections in the similar conditions? Thank you for your quick feedback.

ANSWER:

Thank you for getting back to me.

Yes, I consider heating necessary for successful denaturation and reduction of the samples. We routinely do this in our lab and have for the testing of this antibody. I would also recommend the verification of your sample transfer using the ponceau red dying of your membrane. For further details please see our western blotting guide at the following link;

http://www.abcam.com/assets/pdf/protocols/WB-beginner.pdf

Please refer to section F for details of electrophoresis.

Please get back in touch with me should this not improve the results that you have been obtaining.

I'm sorry to tell you, but the recommended protocol, namely to block with 3%BSA and the overnight incubation, does not give the expected result. I have to admit that the modifications give no better results in compare to milk blocking, but I have got a strong signal of the background including the molecular marker, and no distinguishable detection of the beta actin. Practically I've got a signal of al proteins loaded on the gel.

Best regards

ANSWER:

Thank you for getting back to me. I am sorry to hear that you have been unable to detect beta Actin using this antibody despite my recommedations to your protocol. Further to closer examination of your protocol I would appreciate it if you could tell me whether you heat your samples prior to electrophoresis in the disociation buffer. This is an essential step for correct protein separation.

Thank you for your feedback. Once again I appreciate your patience.

DESCRIPTION OF THE PROBLEM No band, no detection

SAMPLE THP1 total cell lysate in Disagregation buffer(DB) HEK 293 total cell lysate in Disagregation buffer(DB

PRIMARY ANTIBODY beta Actin antibody [mAbcam 8226] (HRP) - Loading Control (ab20272), 1:5000 and 1:1000 in 5% milkpowder in TBST for 1h at room temperature

DETECTION METHOD ECL for 1 min to 1h

POSITIVE AND NEGATIVE CONTROLS USED I use HEK293 cell wich were transiently transfected with ABCG1 protein, for WB I load 25ug of total cell lysate on 7,5% SDS gel, and the PVDF mambrene was stain with mAb against ABCG1 1:100 dilution in 5% milkpowder of TBSTween +2mM Na-azid for 1h, then wash 3 times 15 min with TBSTween, stain with secondary antibody antimouse HRP (Jackson) 1:10000 in 5%milkpowder in TBST for 1h, room temperature. wash 3 times for 15 min than use ECL for 1 min.

ANTIBODY STORAGE CONDITIONS -20 C degree, in 10ul aliquots

SAMPLE PREPARATION DB buffer + sonication, no heating

AMOUNT OF PROTEIN LOADED 75 ug protein/lane

ELECTROPHORESIS/GEL CONDITIONS Reducing gel 7,5% SDS Poliakrilamid, 80mA 20 min., 130mA 100min.

TRANSFER AND BLOCKING CONDITIONS Transfer buffer with 4% metanol, 25mM TRIS, 192mM glicin blocking in 5% milkpowder in TBST over night, with 2mM Na-azid

SECONDARY ANTIBODY no secondary

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? use different dillution of the antibody

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. Our beta Actin antibody [mAbcam 8226] (HRP) - (ab20272) is a popular antibody that we have received relatively few complaints about. Every batch of this antibody is tested and quality controlled in house. The results that you have been obtaining are indeed alarming; especially in view of the fact that you have been loading 75ug of protein.

I have read your technical questionaire and I have a few comments. In view of the valuable feedback that our customers have provided us with I would like to suggest that you try applying this antibody using BSA as a blocking agent. We frequently find that changes to the blocking agent result in cleaner, more prominent results. Therefore by performing an overnight incubation of the antibody supplementing 3% BSA instead of milk I feel that you would obtain more favourable results.

Should this not lead to an improvement in the results that you have been obtaining please back in touch with me and I will arrange for a replacement vial to be shipped (provided this antibody was purchased within the past 90 days).