I am working with suspension cells, JY cells in particular. I have been using hypotonic lysis (buffer components: 10 mM Tris-HCl pH 7.4) to attempt to obtain cytosolic and membrane protein fractions but am having difficulty in doing so.
After incubation with hypotonic buffer, I do observe the cells start to swell up, and after rounds of centrifugation I also observe some membrane ghosts from the cells. Upon western analysis, I still find alot of cytosolic proteins in my ”membrane” fraction but not much membrane proteins in my “cytosolic” fraction (which is correct). Does anyone have an explanation for this or any suggestions I could make? I am currently using detergent-free methods because I will be doing mass spec analysis after lysis.
I'd suggest adding a physical disruption step, such as sonication, if you don't have one in the procedure already. You could also try using a glass homogenizer with a tightly-fitting pestle. The membranes will most likely be broken up into little pieces by this procedure.
Adam B Shapiro hey Adam thanks so much for your response. after sonication is it sufficient to use benchtop centrifugation to obtain membrane protein fractions or is it necessary to use ultracentrifugation?
You could start with a low-speed spin to remove the dense nuclei and unbroken cells. I would then use ultracentrifugation to be sure of maximizing yield. 1 hour at 100,000 g should do it. The membranes can then be fractionated according to organelle origin by sucrose density gradient ultracentrifugation.
You can get rid of detergents after cell lysis before mass spec analysis. There are options such as S-trap and mainly FASP. Use urea to disrupt formed micelles and remove detergent monomers by serial ultrafiltration and simultaneous buffer exchange application.
Do not forget, that you can get high recovery protein extraction in particular to membrane proteins by harnessing detergent ingredients. Alternatively, mass spec compatible (acid cleavable) detergents can also be evaluated during lysis such as Deoxycholate (relatively cheap)...
Good luck
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