I have sonicated six HeLa-cell samples in RIPA lysis buffer (prior inc. on ice, 15 min). For one of these samples, the sonication was unsuccessful. My question is what factors should be taken into consideration when trouble-shooting? This is for a student project, so the important thing is throwing every possible cause out there! Thank you.
Dear Amanda!
Please you look at this article:
Article An Analysis of Critical Factors for Quantitative Immunoblotting
http://tools.thermofisher.com/content/sfs/brochures/1601757-Cell-Lysis-Handbook.pdf
Sonication
https://openwetware.org/wiki/Griffin:_Ultimate_Immunoprecipitation_Guide
As for myself who have been extracting variety of proteins from the tight smooth muscle tissue I do not believe in sonication in lysis buffer. I consider consider fragmentation (homogenization) and solubilisation in a 8 M urea buffer to be more effective if the latter is acceptable for your experiments. Most effectively one can remove the urea even from much diluted samples by 10% TCA precipitation and pelleting with centrifugation. Traces of the urea and/or TCA can be removed by rinsing of the tube wall with water. Of course dissolving in the urea means denaturation. Re-naturation is often possible especially for small proteins. This method can be used for 40 or more fold concentration of also for the larger protein but these have to be dissolved again the tiny volume of the same urea buffer.
For fragmentation of hard tissues use tiny glass-glass homogenizes.
I hope that you can use something from these suggestions. For more exact one I would need to know more about your procedures and their aims.
Best regards,
Apolinary
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