Hi All,
I was recently isolating proteins in interstitial fluid from tissue cells. To do that, I firstly used PBS (+proteinase inhibitor) to homogenize the tissue. And I spin the cells down at 12k rpm to get the supernatant, which then it is the protein from interstitial fluid. Then I add in lysis buffer (RIPA) to lysis the cells and get the protein inside the cells.
I'm a bit concerned that the one step of high spinning could break my cells, which makes the result confusing. So my question is whether this method makes sense and whether hight speed spinning breaks cell membrane?
Thank you all in advance!
Are you not also concerned that some cells could be broken during tissue homogenization?
Adam B Shapiro Fair point. I have that concern too. I noticed even just ordinary grinding procedure could cause 5% cell death. We used VWR pellet mixer in this experiment.
Dear researcher
These links are useful:
http://www.ufrgs.br/biofisica/Bio10003/Centrifug.pdf
https://www.ncbi.nlm.nih.gov/books/NBK26936/
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