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I received your suggestions for the WB assay. We use that dilution of antibodies because was the recomended dilution for the product but we did not try another dilutions because we didn´t have any signal little or too much, likewise, if we used more than 5 ug in the electrophoresis the protein is too hard to denature and inespecific bands will appear that might impair the results; we used also 5% milk, but there were no diferences and the primary antibodies for Pv12 worked fine with both. Due to all of this, in the lab´s meeting the conclusion was that we prefer to make the change of the antibodie to the reference mentioned in the last e-mail: AB24859, I hope you understand our reasons.
Asked on Feb 13 2012
Thank you for the reply.
The recommended dilutions on our datasheets provide a concentration to begin with, however the optimal dilution does have to be determined by the end user. Therefore I strongly recommend that you try various concentrations of the primary antibody, as there is still a good chance that this antibody will work for you, if you can identify the optimal dilution.
Also, I understand that you are worried about high background but could you try and increase the concentration of the protein loaded to 10ug.
If after you have tried the above suggestions you are still not seeing a signal from ab109444, then I would be happy to refund or replace the antibody.
Please let me know if there is anything else I can help you with.
Answered on Feb 13 2012