Question (18519) | Anti-ABCA1 antibody (ab7360)

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Question

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.

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