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Here’s a quick review of what I do:
Cells tested: C2C12 myoblasts AND primary mouse myoblasts
Dilutions & Times:
SATB2 primary Ab (abcam) 1:2500 in 5% Milk-TTBS Overnight at 4C rotating platform
Secondary Ab anti-rabbit 1:5000 in 5% Milk-TTBS for 1hr at RT on shaker
Sample Preparations: Whole Cell lysis protocol, using RIPA buffer; protein amount loaded = 75ug, 10% SDS-PAGE gel, transferred to membrane, blocked in 5% Milk-TTBS solution for 1hr
Basically it’s a straightforward type of thing. In the past I’ve had good results from the SATB2 antibody from you guys, it’s just this recent vial has shown me nothing at all on the developed film. I know my other probes work just fine; it’s just SATB2 that doesn’t work.
Asked on Mar 27 2012
Thank you for providing the details of your protocol.
Have you tried using the primary antibody at a 1:1000 dilution? Because this is a polyclonal antibody, there may be some batch to batch variability. Our internal QC will only pass this antibody if it gives good signal at the dilution stated on our datasheet (1:1000). While the lot you had previously purchased may have been a particularly strong batch and worked at 1:2500, I would recommend using more primary antibody if you are not seeing any signal.
If you would like to try a new batch, I will be happy to send you one as a free of charge replacement. Unfortunately, I was unable to locate a record of your order in our system. Did you by any chance purchase this antibody through our Canadian distributor Cedarlane? If so, can you please tell me the approximate day of purchase so that I can locate the correct order? Once I have found your order I will be happy to send you a free of charge replacement from a new lot.
Answered on Mar 27 2012