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Pippuri 01-05-2009 10:26 PM

Question regarding subcellular fractionation
 
Hello, I have been struggling with one of this experiment for awhile and any input/suggestion is highly appreciated! :notworthy:

I am trying to perform subcellular fractionation to see whether my protein of interest is translocated to plasma membrane in response to different growth factors. So, this is the thing-- from literature, it seems really straight forward and easy, but I have tried different version of the protocol and none of them really worked well in my hands. I also searched through discussion posts looking for tips, but still, I can't get any result...

First question that I have is-- in order to obtain decent amount of protein for downstream immunoblotting analysis, how much cells to start with? I am using both CHO cells and COS-7 cells-- is 1x10 cm plate ~90% confluent enough??

Second, I tried with 250mM sucrose, and stepwise centrifugation (1000xg 10000xg and 100000xg), but the thing is I really don't know the visible floating white thing is normal during the first 2 centrifugation... And I hardly get any pellet after the 100000 xg step... What is going on there? Can someone who with experience let me know the exact protocol in detail so that I can practice couple times to make sure I get decent Akt translocation as a control? (so far I either get nothing, or have Akt in both - and + stimulation... which is really depressing)

Third, I also tried this sucrose cushion method-- I found one of this paper pretty much has this details... but the thing is I can't see the "interphase" which is supposed to be the plasma membrane fraction after ultracentrifugation... How come???

I really need it to work in order to graduate... Please please please...

Many thanks!


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