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Asked on Sep 24 2012
Thank you for contacting us. I am sorry this antibody is giving no signal in WB.
Have you validated your transfer for proteins around this MW? I would recommend performing a Ponceau stain to double check that the protein is present on the membrane because the run and transfer conditions you are using are generally more suited to smaller proteins.
It may also help to reduce the concentration of blocking agent in the antibody diluents. Milk should be kept to a concentration of 1% with the primary antibody and removed from the secondary antibody buffer. It is possible that the extra protein may be competing with the antibody and thereby inhibiting the binding.
How long was your exposure time? Have you tried overexposing the blot to see if there are any background bands detected? How long were your wash steps? Washes should be kept to 3x 5 minutes to prevent a reduction of signal.
I hope this helps to improve your results, if not, please let me know and I will be happy to help you further.
Answered on Sep 24 2012