I have used PBS with protease inhibitors as lysis buffer to extract proteins from mamalian tissue,i choosed this buffer that not contain any detergent affect on my results on mass spectrometry.Is it possible to use PBS with protease inhibitors as lysis buffer for protein extraction from mammalian tissue?an paper can support this protocol?
Hello Eric, hope all's well.
To lyse mammalian cells, you must disrupt the cell membrane(s) to release the cellular contents. Should you wish to avoid detergents due to down stream mass spectrometry. Using PBS with protease inhibitors alone will not extract many proteins for analysis in my opinion.
I would recommend a chaotrope like 8M urea in 20mM HEPES buffer (plus any inhibitors you wish to use) in addition to sonication. You can then centrifuge to remove the cellular debris (including DNA/RNA) and then leave only your proteins for further analysis. You can then use an enzyme like LysC to digest your protein (which is tolerant of high urea content) and then choose to dilute the solution to a lower concentration of urea (
Many thanks Peter.I alreaday used only PBS with protease inhibitors,i just compare methods,and i could not find any paper support my protocol,thats why im looking for paper used only PBS with inhibitors to cited and told the people in the defense that i used this protocol depends on this paper
Dear Eric
I used Tris-HCl with sucrose without any protease inhibitors but perform all procedures on ice and disrupt the cells under liquid nitrogen by mortal and pestle.
Our 2-DE proteome maps were very good.
You can find it in ELECTROPHORESIS 2014; 35 (23), 3331-3338.
Hi,
when I was trying to use mass spectrometry, I also had the same problem like not to use any non-ionic detergent for lysis. But u can use ionic detergent (such as sodium dexoycholate) to lyse the cells as they are compatible with mass spec. Ihave used 7M urea with 1% sodium deoxycholate to solubilize the contents in the cells.
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