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Thanks for your quick reply! I incubate membrane in PAD4 antibodies (1:1000) at 4 degree, overnight. The next day, wash with PBS-tween 5 min,X3 times, incubate with rb secondary Ab for 2h at room temperature, wash with PBST 5 min X 3, and then develop. I use this protocol for other antibodies and they work well. Thanks for passing my comments to the relevant team, and hopefully in the future I can get this Ab that's guaranteed to work with mice.
Asked on May 10 2012
Thank you for your reply.
There are a few variations that may work:
1 - Depending on the blocking agent you are using, you could try using 5% BSA, as this may help reduce the background levels that you are seeing.
2 - If you are seeing the band of interest, but also getting high background, then using a lower concentration of the antibody may be helpful in reducing background..
3 - Another, way to decrease background is to use ab126587, our https://www.abcam.com/10X-Blocking-Buffer-ab126587.html, but I would only recommend that if using the BSA does not prove to be successful.
Please let me know if there is anything else I can help you with.
Answered on May 10 2012