Why different mammalian cell lysate preparation methods? - (Jul/19/2007 )
Hello
I'm currently studying transcriptional regulation and am doing western blotting for both nuclear/cytosolic proteins from HepG2 cells using 23G needle shearing method to extract my total cell lysate.
However, I was told that many others usually use ionic methods (like RIPA buffer etc) to extract TCL, instead of mechanical force such as 23G needle, an established method in our lab - passage cell pellets through 23G needles 10 times in SDS sample buffer (with Tris, glycerol & b-mercaptoethanol only), then boil at 100'C for 5 minutes before centrifuge at high speed at 4'C to collect supernatant (TCL!?)
Can anyone please tell me why different preparation methods and how to choose a particular method? Is it cell line dependent? 
Cheers & Thanks a lot in advance!
generally it's not cell-line dependant. You may play with detergent concentration, and glycerol maybe.
Seems you're in laemmli for your total cell lysate... and do western blot. Then how do you quantitate your protein concentration ?
Hi Fred33,
I first precipitate an aliquot of each sample by TCA then do a Lowry or BCA assay.
The thing that concerns me is - sometimes I don't get very good shearing, which is evident from the "protein glob" (after heating and centrifuge) that happens at times, even though I always adjust my sample buffer (yes its laemli!) to different cell numbers accordingly... and of course I make sure I do 10x exact through the needles.. any idea why this might occur?
Can I solve this problem by playing with the detergent (SDS) concentration? I've heard to increase glycerol to freeze cells for longer storage..
I first precipitate an aliquot of each sample by TCA then do a Lowry or BCA assay.
The thing that concerns me is - sometimes I don't get very good shearing, which is evident from the "protein glob" (after heating and centrifuge) that happens at times, even though I always adjust my sample buffer (yes its laemli!) to different cell numbers accordingly... and of course I make sure I do 10x exact through the needles.. any idea why this might occur?
Can I solve this problem by playing with the detergent (SDS) concentration? I've heard to increase glycerol to freeze cells for longer storage..
You said that the sample buffer you used for your lysate does not have SDS? If you just use mechanical mean (passage through needle) to lyze cells, then I am afraid that the 23G may not be small enough. That could be the cause for the 'not very good shearing'. When you need to infect cells into mammals, such as mice or rats (especially in studying cancer), the needle we use is 25-23G, so cells should not be broken in that case. We have found that even 25G may still not sufficient (you can just pass cells through the needle 10x and then take a sample, mix with trypan blue and look under microscope to see if cells have been broken properly). We are using 29G currently, or the dounce.
well it happens to me the fact that the aliquot in laemmli buffer forms a pellet. I dilute it with little amount of running buffer and it helps loading.
for total cell lysate, i posted a protocol here
I first precipitate an aliquot of each sample by TCA then do a Lowry or BCA assay.
The thing that concerns me is - sometimes I don't get very good shearing, which is evident from the "protein glob" (after heating and centrifuge) that happens at times, even though I always adjust my sample buffer (yes its laemli!) to different cell numbers accordingly... and of course I make sure I do 10x exact through the needles.. any idea why this might occur?
Can I solve this problem by playing with the detergent (SDS) concentration? I've heard to increase glycerol to freeze cells for longer storage..
You said that the sample buffer you used for your lysate does not have SDS? If you just use mechanical mean (passage through needle) to lyze cells, then I am afraid that the 23G may not be small enough. That could be the cause for the 'not very good shearing'. When you need to infect cells into mammals, such as mice or rats (especially in studying cancer), the needle we use is 25-23G, so cells should not be broken in that case. We have found that even 25G may still not sufficient (you can just pass cells through the needle 10x and then take a sample, mix with trypan blue and look under microscope to see if cells have been broken properly). We are using 29G currently, or the dounce.
Well, SDS is in the laemli buffer...without it of course lysis won't occur. What kind of lysis buffer do you have when u use 29G (or dounce)? and what are cell lines?
ps fred33, thanks for the thread, its very useful~
by the way, do you know why protease inhibitors are omitted in the laemli buffer? our lab's protocol doesn't seem to use them when making total protein, but we do when extracting nuclear proteins... WHY!!? anything in the laemli make proteins invulnerable to proteases?
hi,
because of SDS ofcourse!!! in laemli buffer all proteins are in peptide form, in this confirmation i do not thing we need to use some protease inhibitors.
rgds
by the way, do you know why protease inhibitors are omitted in the laemli buffer? our lab's protocol doesn't seem to use them when making total protein, but we do when extracting nuclear proteins... WHY!!? anything in the laemli make proteins invulnerable to proteases?
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