Anti-beta Actin antibody - Loading Control (ab8227)

WB, Ab worked before
new lot (GR62165) shows various non-specific, fuzzy bands (around 60 and 100 kDa), but none at 42 kDa
293T cells, 5-7 ug protein loaded
Ab: 1/100, 1/500, 1/1000
block: 5% milk
2nd: works fine

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Hello, here are the answers to your questions:

-I don't think there is a possibility of the gel being overloaded as we used different total protein concentration, as low as 25ug and still the problem persisted. 100ug is tolerated well by the type and thickness of gels we prepare. Also, we checked the transfer with Ponceau Red and it was good in all cases.

-The material used was total lysates from rat cortex tissue, prepared with very standardized protocols we have at the lab, which work well for all other antibodies that we are currently using for Western blotting.

-What we want to see with the antibody is nitrotyrosine in all proteins in general, we will then perform IP to see whether our protein of interest (cytosolic) contains nitrotyrosine residues.

-The samples have not been treated to stimulate nitrosylation because we want to determine whether this occurs in an Alzheimer transgenic model which there is evidence that this is the case. We have also been working with aged animals (14 months old) and there is also evidence that nitrosylation increases with aging (published in rats).

-Were these antibodies tested previously in tissue lysates?

-If you have other anti-nitrotyrosine antibodies could you consider the possibility of sending us a few aliquots to test which one is more suitable to our purposes?

Thank you,

I hope we can find a solution to this problem,

ANSWER:

Thank you for getting back to me and for answering to my further questions promptly.

I have been discussing this enquiry with my colleague in the Lab and the methods seem to be chosen all look appropriate ECl, milk block etc, Xray film.

It is a bit surprising that there aren’t backgrounds from the rodents immune system (rat circulating IgGs) so you might wish to change the secondary HRP conjugate.With issues like this we are always presuming that there are sufficient nitrated tyrosines in the sample in question to be seen.

Is the level high enough to back up the hypothesis?

This target is a product of the right kind of oxidative stressors and without those there won’t be any modification. To validate this, trying another antibody (rabbit polyclonal) would be a good idea..

To show this antibody works we can use a positive control – add peroxynitrite to the sample – this can be obtained from Millipore. Other donors include SNAP or SIN1 which would create 3-NT.

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

We bought 1 Anti-nitrotyrosine Abcam antibody Ab24496 (received 27-10-2011) lot # GR23989-3which didn't work and was replaced by Ab110282 (received 2-12-2011) lot# GR63357-2. In both cases, the detection of specific nitrotyrosine bands was unsuccessful, the blot appeared dark with white bands or just dark with no bands. We have tried the following conditions for both antibodies:

-Different blocking conditions (BSA 5%-10% and milk 5%-10%)
-Protein concentration (25 ug - 100 ug)
-We used rat and mice cortex tissue
-Western blot detected with Regular ECL and ECL plus
-Detection with autoradriographic film and Storm phosphoimager
-Concentration of 1ry antibody 1:100 - 1:5000
-Concentration of 2ry antibody: 1:5000-1:50 000
-Our secondary antibody is working well with other primary antibodies

If we cannot get reimbursed, can we get this antibody instead: Anti-beta Actin antibody - Loading Control (ab8227)

Thank you

ANSWER:

I am very sorry to hear that both antibodies did not work properly (ab24496 and ab110282).

I can offer you a replacement vial as you wish but it would be important to find out why two antibodies failed to work.

I have read through again the detailed protocols you kindly forwarded to Abcam and I would like to make the following comments/suggestions:

I understand that rat brain lysates were used in these experiments and 25 ug - 100 ug total protein was loaded onto the gel. It may well be tha the gel is overloaded.

Q1: Could you please confirm if total lysates or cellular fractions were used?

Q2: Are you particularly interested in cytosolic protein or any cellular organelle-related proteins?

Q3: Have you treated the samples to stimulate the nitrosylation of teh protein?

As you can see on the Western blot images (ab110282) purified bovine heart mitochondria was used for testing and characterization.

Thank you for your understanding and co-operation in this matter.

I look forward to hearing from you soon.

Did someone actually report a problem with this batch of antibody? Do you have other lot number that someone tested for sure? Reason why I ordered this was because of its good reputation based on your customers' review. Can I get another batch of beta actin loading control antibody?

ANSWER:

I do not see other complaints for this lot. We do occasionally see reports of poor western results but they do not correlate with any particular lot, and we do not have an explanation for the poor results if the customer's protocol is correct.

Your protocol appears to be correct, if you are confident that the small sample size is adequate. If you are still interested, I can send a vial from a different batch of this antibody. Please just reply to confirm

That is my typo. We got beta actin loading control which is ab8227 and its lot number is GR60183. In my experiment, it is impossible to load more than 1-2 ug of total protein. Since we use the Infra Red visualization system with great sensitivity, as long as antibody quality is good, 1 ug of protein should be good enough. Actually my other antibody worked really nicely.

My tissue of interest is in RIPA buffer. I can't think of anything else than the antibody quality since my other antibody worked precisely.

ANSWER:

Thanks for your reply.

It does seem that this vial of ab8227 is not peforming as expected. If you have purchased this product within the last 6 months or so I would be happy to provide a replacement, credit or refund.

Please let me know how you wish to proceed. In your reply please include your Abcam order reference number or PO number so I can process your request.