In cultured transiently transfected 293T cells there is a small amount of background with the anti-RFP antibody after 1-2 hrs exposure but no bands could be detected at the correct size for the ARF-RFP fusion (˜40kDa. See attached: lanes 1-3 should have expression, with lane 2 having the most protein) . The blot was then stripped and probed with anti-p19 and a band of the correct size could be detected and was induced to even higher degree in the presence of rtTA and following the addition of doxycycline (lane 2; ARF-RFP is driven from a doxycycline- and rtTA-dependent TRE promoter). Thus I am confident that the construct works and the protein is generated in vitro. If used on transgenic mouse tissue samples, there is more background (as observed with many antibodies) but no band corresponding to ARF-RFP.
The lysates were boiled at 95’C for 5 minutes.
Yes, our secondary antibody works (checked against other anti-Rabbit antibodies in the lab).
I cannot find the purchase order number right now but have emailed our accounts team and will get back to you.
I have indeed checked expression under a fluorescent microscope and I see very robust expression in cells transfected with the vector.
I hope some of that info helps. Do not hesitate to contact me if you require more information.
Asked on Jun 28 2012
Thank you for your enquiry regarding ab28664 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
The protocol looks absolutely fine to me. I could offer you either a new vial of ab28664 or an alternative antibody against the same target or a credit note which you can use for future purchase.
Please dolet me know how you wish to proceed with your enquiry. . I look forward to hearing from you soon.
Answered on Jun 28 2012