Reply to: Lysis conditions of the cells/ Wolfgang post

RNA Master Purple.Haze1st at gmail.com
Thu Aug 24 14:13:04 EST 2006


Hello

If you use 0.1% TritonX100 you are permeabilizing such structures as
Endoplasmatic Reticulum, and as you know it is full of proteins.
Actually i am surprised of your protocol. Freeze/thaw then lyse -> then
flash freeze-> ultrasonic.... and all this stuff is just for western?
Are you kidding?
Even for IP it is enough to do freeze/thaw once and take the super, of
course if you are not doing co-immunoprecipitation. With all those
above mentioned steps you can freely reduce your method to just
dissolving the cells in Laemmli, supplied with 20% sucrose and 2%
beta-mercaptoethanol, and sonication.

If you wish to obtain only cytoplasmic fraction why dont you use Dounce
homogenizers with a spherical pestle tip, they destroy everything but
the nuclei. Or some other detergent, that is not as harsh as TX100.
CHAPS, digitonin, saponin.....

Of course, you can do some fractionation if you wish...


A lot of choices


Best regards




>What will I actually solubilize if I treat cells with a lysis buffer
>that contains 1% tx100 as detergent?


>More detailed: Grew hepatoma cells on dishes, washed them with PBS,
>froze them in -80, scraped frozen layer into lysis buffer, pipetted up
>and down a few times, flash froze in nitrogen, thawed in ultrasound
>bath, spun down, I am using the supernatant and kept the pellet. Then
>ran westerns with the supernatant.


>This is a modified procedure where I used to treat adherent cells
>directly with that buffer and the cytoskeleoton and the nuclei remained
>on the dish.


>I assume that I might get something off the membrane and then just have
>the cytosol.


>What will I get if I potter or ultraturrax the scraped off cells?


>Thus, I am looking for some more detailed insights into detergent
>fractionation of cells. Any insights you would like to share?


>Best regards, 


>Wolfgang



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