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Here, I send you my WB condition and the images as attached files.
We purchased anti-UTY antibody within six month. Other two are more than six.
Asked on Mar 06 2012
Thank you for your reply and for confirming the steps of your protocol.
Since these antibodies are Fast Track antibodies, they are only guaranteed to work in ELISA using the peptide immunogen. These are not tested in-house, and therefore not guaranteed to work, in WB with lysates. In addition, 2 of the antibodies, ab36938 and ab38113 were purchased in 2008 so it may be that these antibodies are no longer viable.
The protocol looks okay, but since you're not seeing the bands at the proper MW, you may want to increase the concentration of both the primary and secondary antibodies.
You can also increase the amount of protein loaded. I see that you loaded 2 x 10^5 cells/lane, but we recommend at least 30 ug.
We do not know if these protocol suggestions will help with the antibodies since we haven't validated them for usein WB so we don't have internal protocols to reference. However, I hope this information helps. Since these Fast Track antibodies aren't covered by the Abpromise, it is best to try to augment the protocol to optimize the results.
Answered on Mar 06 2012