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Reply to: Lysis conditions of the cells/ Wolfgang post

Reply to: Lysis conditions of the cells/ Wolfgang post - Protocols and Methods Forum

Reply to: Lysis conditions of the cells/ Wolfgang post - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 08-24-2006, 07:13 PM
RNA Master
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Default Reply to: Lysis conditions of the cells/ Wolfgang post



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



















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Old 08-31-2006, 09:40 AM
Wolfgang Schechinger
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Default Reply to: Lysis conditions of the cells/ Wolfgang post

Hi,

Many thanks for your suggestions!
Actually, I just want the plasma membrane and sometimes the cytosol.
Most of the protocol is a local lab myth that must not be questioned.
The lysates also should be usable for IP, ELISAs and enzymatic assays,
thus the overkill.

Just the sonication step is what I added and I realized that it
probably is not necessary. Normally, the cells are homogenized with an
ultraturrax.

Best regards, Wo

RNA Master wrote:

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