Hello All,
I was wondering if anyone has experience with performing Western blot on mouse ear clippings? We are looking to evaluate the systemic effect of an administered drug on p-EGFR levels across different time points. Our results have been highly inconsistent. It seems freshly homogenized tissue produces the best result, but once frozen and thawed to run again the levels change and are much more difficult to detect. All samples are homogenized in RIPA buffer, supplemented with protease and phosphatase inhibitors, using a bead homogenizer. We have utilized both a Bio-Rad and Invitrogen electrophoresis setup, both with similar outcomes.
Does anyone have a reliable protocol for Western blot on mouse ear clippings that we can try?
Thanks for all your help!
Weigh your ears and match (.0114gm) with RIPA = 114ul Ripa.
Make sure you have 0.1mM DTT, protease, & phosphatase inhibitors in. Scissors homogenize till very fine, then hit with a sonicator once....quick, briefly, on ice. Spin, do your total protein assay using BioRad's DC Assay. Be sure to add solution "S" to "A" component to make it compatible with your homogenizing buffer. Make up multiple aliquotes of your full strength lysate. Never freeze/thaw samples, they degrade each time and protein will be all over the board if you compare a freeze thawed to a fresh.
Good luck!
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