Anti-PRPF8/Prp8抗体(ab79237)

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab79237. I would also like to clarify a couple of details. 1. has the secondary antibody been shown to function appropriately? 2. Are you applying protease inhibitors in the lysis buffer? 3. Are the samples tested with the ab51366 exactly the same? What are the differences between the A, P, NS1, 1-93, 94-239 samples loaded?  I would suggest increasing the concentration of the antibody, we have used 1 µg/ml with this antibody previously (1/500) but I would also try 1/250. I would also suggest incubating the antibody with the membrane overnight at 4°C and reduce the blocking content of the diluent (to 0.1%). I would also suggest using BSA as a blocking agent as this has been used in house to characterise this antibody. Should the suggestions not improve the results, please do let me know, the applications for which you are using it for are using it are covered by the Abpromise and should the suggestions not improve the results. you would be entitled to a free of charge replacement or credit note. I hope this information is helpful, and I thank you for your cooperation.  

I suggest trying anti-ATE1 ab90561, which is a mouse monoclonal, tested for reactivity with human Jurkat but untested on mouse samples. One issue might be reactivity of an anit-mouse IgG secondary with endogenous IgG in the mosue lysate, but this is unlikely to be a problem with 3T3 cells. We do not have another anti-ATE1 rabbit polyclonal, or another lot of ab84636. For PRPF8, I suggest a rabbit polyclonal, ab87433. This is tested against the same cell lines as ab79237 but differs in that it is raised against a peptide from the other end of PRPF8, the C terminus. Please note that PRPF8 is a very large protein, and that efficacy of transfer may be an issue. Were you able to confirm that transfer was successful, for instance with a Ponceau Red stain? If it is not, then another antibody will not help. For large proteins, we recommend PVDF membranes instead of nitrocellulose, removing methanol from the transfer buffer, and including SDS to a concentration of 0.1%. Please let me know what you would like. A credit or refund is also an option.

We have two other antibodies against each of these targets which may give you a better result. However, before sending replacements, we typically collect details of the protocols from our customers before issuing a replacement or credit/refund, in the hope that there may be a simple modification that fix the problem. The western blot protocol for each of these antibodies is standard, and I expect they are similar to what you tried. In brief, 10 ug of lysate was loaded per lane of the gels. After transfer, the blots were blocked with 5% milk, and then incubated with 1 ug/ml antibody, then visualized with ECL. Can you please confirm that this is what you did? If not, please send the details of your protocol. I do not know if expression of ATE1 in these cell lines is supressed; a quick search online showed some mentions of transfected 293T and 3T3, but none of endogenous expression. The antibody ab84636 was tested on HepG2, and another antibody, ab90561, was tested on Jurkat. Do you have evidence that 293T and 3T3 express ATE1?