Direct Cell Lysis in Laemmli - (Oct/15/2008 )
Hi, I am kind of sick and tired of doing cell lysis with Triton, etc, then always having to do Bradford or the like to get protein concentration before I load on the gel prior to blotting.
Does anyone know tips and tricks for direct cell lysis in Laemmli buffer? I know this can be done, I just don't know the following:
1) How many cells to load (I usually load 10-30 ug protein in each well of a lysate)
2) How much protein can I expect from a certain number of cells, average?
3) How long do I boil? Spin?
4) How do I not melt my tubes for a long boil like this and how do I not pop the caps and lose sample?
Thanks for the replies. I don't do much protein work (no one in my lab does) but I need some quality westerns!
-jtotheizzoe-
QUOTE (jtotheizzoe @ Oct 15 2008, 02:06 PM)
Hi, I am kind of sick and tired of doing cell lysis with Triton, etc, then always having to do Bradford or the like to get protein concentration before I load on the gel prior to blotting.
Does anyone know tips and tricks for direct cell lysis in Laemmli buffer? I know this can be done, I just don't know the following:
1) How many cells to load (I usually load 10-30 ug protein in each well of a lysate)
2) How much protein can I expect from a certain number of cells, average?
3) How long do I boil? Spin?
4) How do I not melt my tubes for a long boil like this and how do I not pop the caps and lose sample?
Thanks for the replies. I don't do much protein work (no one in my lab does) but I need some quality westerns!
Does anyone know tips and tricks for direct cell lysis in Laemmli buffer? I know this can be done, I just don't know the following:
1) How many cells to load (I usually load 10-30 ug protein in each well of a lysate)
2) How much protein can I expect from a certain number of cells, average?
3) How long do I boil? Spin?
4) How do I not melt my tubes for a long boil like this and how do I not pop the caps and lose sample?
Thanks for the replies. I don't do much protein work (no one in my lab does) but I need some quality westerns!
geez, do you believe me if I say I just logged in to ask the same question?
my friend lyses HeLa cells directly in 100-200ul SDS-PAGE buffer without protease inhibitors and boils them.....she says this way she won't lose much protein and her samples are purer. she boils for 10-15 mins. tubes won't melt. 30ug+ is sufficient in each well.
i normally use RIPA lysis buffer and then use the lysates for my SDS-PAGE, so I'm also wondering if direct lysis is a recommended method or not ?!
-Curtis-