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First batch worked on a couple of Western blots and stopped working. Initially I thought the antibody got degraded by protease, etc. so I ordered another batch and never got Western blot working ever since. I'd like a replacement for another product (ab57553). Amount of protein loaded: 10ug HEK lysate Blocking: 10% dry milk + 5% normal goat serum in TBST Detection method: ECL and Licor infrared 7.5% gel ran at 130V, transferred at 80V 400mA 5 hours Primary antibody conditions: 1:1000, 1:500, 1:250 for 2hr up to o/n Secondary: rabbit HRP from Amersham 1:10000, 1hr RT washing 3 times in TBST
Asked on Jul 06 2012
Thank you for contacting Abcam.
I am sorry about the issues you are having with ab114126 in western blot. The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot on human samples . If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.
I would just like to ask a few more questions:
1 - Can I ask how the antibody was being stored? Was if frozen at -20C or kept at 4C?
2 - You block in 10% dry milk + 5% normal goat serum, have you tried just using 5% milk? I ask as the blocking agent you are using is different than what is normally used.
3 - When you did get a signal using the antibody, was it at the correct/expected molecular weight?
Also, would you be able to provide me with either the Abcam order number or PO# that was used in the purchase of the antibody,that would be very helpful.
I look forward to your reply.
Answered on Jul 07 2012