|Register||Search||Today's Posts||Mark Forums Read|
|Protein Science Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.|
| ||LinkBack||Thread Tools||Display Modes|
Question regarding subcellular fractionation
Hello, I have been struggling with one of this experiment for awhile and any input/suggestion is highly appreciated!
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...
|fractionation , question , subcellular|
|Thread||Thread Starter||Forum||Replies||Last Post|
|Trouble with subcellular fractionation||Tiffy||Protein Science||0||04-26-2008 04:30 PM|
|subcellular fractionation and mAbs||Morwenna Porter||C Elegans Forum||0||04-03-2007 10:08 PM|
|A question about studying plant gene subcellular localization byusing eYFP||Chan Kam Ho||Arabidopsis and Plant Biology||2||01-30-2007 11:32 AM|
|Sci.chem FAQ - Part 1 of 7||Bruce Hamilton||Chemistry Forum||0||01-15-2004 08:06 AM|