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Here are the answers for your questions,
1)Yes, we transfected human AF9-Flag into 293T cells
2)2*loading buffer was used to lysate our samples and about 10ug protein
was loaded per lane.
3)5% non-fat milk in PBS for 1.5 hour
4)The primary antibody (1:500, over 8 hours at 4 C)
5)Secondary antibody: Goat anti rabbit HRP (1:7000, 1.5 hours at room
6) The antibody was ordered on May 30 with order number ***.
Let me know if you have more questions, :-)
Asked on Jul 31 2012
Thank you for your reply and for providing these details of your protocol. I agree that the background bands you detected should not be present using your protocol. The only suggestion I could make is that because AF9 is a nuclear protein, you may see clearer results using a RIPA buffer for lysis and loading at least 20ug of sample per lane. If you are unsatisfied with your results, I will be happy to offer you a free of charge replacement antibody or a credit or refund. Please let me know how you would like to proceed and I will be happy to assist you further.
Answered on Jul 31 2012