Anti-ABCA1 antibody (ab7360)

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE human liver membrane, liver microsome and liver postnuclear lysates PRIMARY ANTIBODY 1:1500 in 3% milk TBST DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED no ANTIBODY STORAGE CONDITIONS 4 degree SAMPLE PREPARATION sucrose, tris, et. beta-meceptoethonal at room temperature 20minutes or DTT 37 degree 30 minutes AMOUNT OF PROTEIN LOADED 40microgram ELECTROPHORESIS/GEL CONDITIONS 3-6% SDS PAGE or 3-8%SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Tris-Glycine buffer with or without SDS, 10 or 20% methonal Blocking: 5% milk TBST SECONDARY ANTIBODY Donkey anti rabbit F(ab)'2 fragment HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Others I follow the protocol on Another companies datasheet for ABCA1 antibody.

ANSWER:

I'm sorry to hear you are having a problem with ab7360.

I would like to suggest the following modifications to your protocol:

The recommended dilution range for this antibody is 1/500 - 1/1000. You may be using it at too high a dilution.

I did not see your incubation conditions with this antibody from your enquiry. Since you are seeing no signal, I would suggest removing the milk from the primary antibody solution and incubating at 4C overnight. The milk in the primary antibody solution can overblock the membrane, preventing the antibody from binding to its target.

I would continue incubating the sample in loading buffer for 30 minutes at 37C. This protein can form aggregates, preventing migration into the gel matrix.

For how long did you block? If you have blocked for longer than one hour, you may wish to reduce this step to 1 hour, or even 30 minutes.

Did you check (e.g. Ponceau stain) to see that the transfer was efficient?

Please let me know if this helps and do not hesitate to contact us for further advice.

Thank you for your prompt reply. In answer to your questions, I used the antibody at both 1 in 1000 and 1 in 500 dilutions, for both overnight and 1 hr incubations at 4 oC, blocking and dilution buffer for the antibody in all tests was PBS + 0.1% Tween, I have tried the secondary alone and it was clean, and offer course borrowed the ab18180 from a collegue and got the desired results. The antibody was aliquoted and stored in the freezer. I hope this helps and look forward to hearing from you.

ANSWER:

Thank you for your reply and the details of the experiment. You do not seem to mention any blocking agent in the experiment and we have found that it is important to block the membrane in 5% milk for 1hr at RT, washing in TBST and adding the antibody diluted in 3% milk in TBST.

We have noted on the datasheet that "Additional non-specific bands are seen at lower molecular weights, but do not interfere with the ABC1 signal" but if you still do not see a band at the correct MW with a blocking step please do not hesitate to contact me, providing your order details and I will arrange a replacement of the other antibody if the antibody was purchased in the last 90 days,

I recently purchased ABCA1 antibody (ab7360) for use with RAW cell lysates and have had very disappointing results, with no bands of the size expected, however use of your ABCA1 (ab18180) antibody gave very good specific results with the same lysate. Could you suggest why this would be the case and if possible replace the ab7360 for ab 18180.

ANSWER:

I'm very sorry to hear you have been experiencing problems with ab7360. The fact that you are having good results with ab18180 rules out that the problem is due to your samples, and I would appreciate if you could tell me more about the conditions you have used ab7360 in, as one or more will be responsible for the problem, or the antibody is damaged.

-what dilution have you tested? -what incubation time have you tested? -what blocking buffer have you used? -what antibody dilution buffer have you used? -have you tried the secondary antibody alone; or with other primary antibodies? -how did you store the antibody?

If you could please provide your order details I will also look at possible shipping delays which would indicate a damaged vial. If the problem is due to the antibody and it was purchased in the last 90 days I can certainly offer you a replacement vial of ab18180.

I look forward to hearing from you to resolve this matter, thank you in advance for those details,

hello: i have already bought this ABCA1 antibody, and I've read the application of this product. It doesn't mention that if it can be used for ELISA. Can I use this antibody to do ELISA? Shall I lyse cells by WB lysis at first? Or could you send me some ELISA protocols for this products instead? I'm looking forward to your reply. Best wishes and warm regards!

ANSWER:

Thank you for your enquiry. To our knowledge, this antibody has not yet been tested for application in ELISA. It doesn't mean that the antibody won't work, we just don't know. We have some very nice ELISA protocols available on our website which should be helpful for you. To find these protocols, you can click on the "protocols" tab located on the online product or by clicking on the "resources" menu located on the Abcam homepage.

Please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image).

Please contact us again if you have any additional questions.

Thank you for your kind help. I received an inquiry about your product, #ab7360; ABCA1 antibody

Would you please let me know how to prepare 'postnuclear lysates' which is noted on the recommended protocol for WB with ab7360, http://www.abcam.com/index.html? pageconfig=protocols&pid=282&intAbID=7360&strTab=protocols&mode=prot

If you have any information, please let me know.

ANSWER:

We do not have an exact protocol but I do have the following information that should provide useful:

Postnuclear samples means that the cells are broken open either by sonication or dounce homogenization and the nclei are removed by low speed centrifugation. The resulting supernatant is called postnuclear. This supernatant contains most of the cytosolic and membrane proteins without containing nuclear components.

Most of the references listed on the datasheet(that are doing westerns) list a brief description of how they obtained postnuclear lysates but it appears that none are highly detailed. I hope this helps.