IP from frozen protein extracts - (Aug/11/2006 )
Hi
As anyone tried to do an IP using protein extracts that were frozen?
My guess is that results depend on the proteins of interest but does anyone had a good experience with this?
Thanks
We freeze all protein samples before we plan to do anything with them including IP.
So do I.
I never had any problem.
what kind of lysis buffer do you use?
Do you add glycerol to your protein extracts after lysis and before freezing them to protect proteins?
Do you add glycerol to your protein extracts after lysis and before freezing them to protect proteins?
yes I used glycerol
0.1% triton X-100
10% glycerol
NaCl 150 mM
EDTA 2 mM
PMSF 1mM
roche protease inhibitor cocktail
Na3Vo4 1 mM (when working with phosphorylation)
in tris-HCl pH8.0 20 mM
No, we don't add glycerol.
Our extraction buffer.
TBS-Triton extraction buffer
50 mM Tris ph 7.5
150 mM NaCl
0.1% Triton X-100
10mM NaF
Add DTT to 1.5mM and PI to 1X immediately before extraction
what kind of lysis buffer do you use?
Do you add glycerol to your protein extracts after lysis and before freezing them to protect proteins?
yes I used glycerol
0.1% triton X-100
10% glycerol
NaCl 150 mM
EDTA 2 mM
PMSF 1mM
roche protease inhibitor cocktail
Na3Vo4 1 mM (when working with phosphorylation)
in tris-HCl pH8.0 20 mM
----------------------
Do you need to add PMSF with roche PI cocktail tablet???
Hi Missele,
I also wanted to know the same thing.
Do you add PMSF as well.
I think PMSF is a cisteine protease inhibitor that is different from the ones present in the protease inhibitor cocktail from Roche.
If you have only 0.1% triton X100 I assume you lyse cells by sonication or other method right?
How do you do that?
And how do you freeze the protein extracts in dry ice, liquid nitrogen and keep at -80C ?
Thanks
Actually, I think it's redondant.
PMSF is a serine protease inhibitor, and I used to use it in combination with leupeptin and others inhibitors.
Then I found that the complete TM tablets were easy to use. But I don't know why I kept PMSF, it should be useless because the complete tablets contain also serine protease inhibitors. 
Well, maybe it's not useless the use of PMSF because you are not the only group that does that. I often see published papers where they use PMSF and complete protease inhibitors.
Can you please answer my other question?
How do you lyse cells? and do you freeze lysates?
Thanks