I have been trying to isolate protein from thioglycolate-elicited peritoneal macrophages. After harvest, I plate cells at ~1-2mil/well, wash out non-macrophages the next day, and wait till confluency to isolate protein using a confirmed protocol (50-100ul RIPA + protease inhibitors). It always seems that I have plenty of protein (viscous lysate), but BCA quantitation shows very little protein and WB show no protein banding. I believe the error is in the protein harvest protocol, but not sure. Help?
Viscosity could be attributed to nucleic acids. Ultrasound treatment/extraction could improve protein extraction efficiency.
2x10^6 cells per well.. what is the area? resulting plating density?
Inflammatory cells could be toxic for themselves so media/plating time could account. If the mentioned amount corresponds to the total number of peritoneal cells it could be too low for mice after thioglycolate (strain dependent).
What is the amount of protein loaded in each WB band?
Do you have positive control for staining and did you checked electrophoretic separation by Ponceau Red staning f.e.?
As long as you have viable cell culture, there should be a good quantity of protein given that the cells lysed perfectly. First, I would check the efficacy of the RIPA buffer you are using. Viscosity is attributed to nucleic acids and at the same time an indication of partial cell lysis. I would add an extra volume of lysis buffer to see if the lysate is getting any better.
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