protein extraction from stable mammalian clones - (Mar/16/2006 )

Dear all,
I am kind of new about these kind of protein thing from mammalian cells. anyone can give me some ideas? what kind of lysis buffer you use and what technique to break the cells? sonication?

what is th econdition? thanks a lot

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hi
different protocols exists.
I have three different categories of protocols.
-membrane protein (i'm not in touch with these so i hope someone else may give a functionning protocol )
-total cellular extract
-proteins from cytoplasm and proteins from nucleus in separate tubes

General procedure is to add protease inhibitors and to work at 4°. I use a lysis buffer and pipett gently up and down. Hold cells in this buffer allows an efficient cell lysis.
I can send you these protocols. (i'm sure to previously posted them on the bioforum, but where in the 1700 posts i did? sending is quicker).

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Thank you fred-33. sos so helpful. basically I am not sure where my protein is, I am sure that in nucleas mostof my protein is there, but not sure that whether it also appears other place. could you kindly send me the protocal? thank you sos so much. hehe

by the way, do you use the RIPA bufffer.? I read couple of paper and they use this buffer.

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hi
i don't use a ripa buffer but a np40 based one

Full proteins :
*use 300µl of lysis buffer for about 5 millions of cells
*incubate cells + lysis buffer 10' on ice
*Centrifuge 14000 rpm 30' 4°C
*discard the pellet
*the extract is ready for quantification and more analysis

sample buffer :

NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160µl EDTA 250mM)
NP40 1% (2ml NP40 10%)
Tris HCl 50 mm pH 7.5
ddH2O qsp 20ml

just before use add for 1ml of buffer :
-5µl DTT 1M (to get 5mM)
-1µl PMSF 100mM


Nuclear/cytoplasmic proteins :
this one works in my hands
But sometimes, i get par of pellet in the eppendorf. So i decided always centrifuge max speed (at least 20 000g) for 5' at RT before the quantitation.

Wash cells with cold PBS twice
Add 4ml PB and scrap them (or trypsinization)
Pellet by 5’ 1000rpm 4°
Transfert in eppendorf
Pellet 2000 rpm 4° 5’

Resuspend pellet in twice the pellet volume of cold A buffer supplemented by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M

Add 0,05%NP40 after added Abuffer in all samples. Pellet 15’ 2500g 4°

Supernatant contains cytoplasmic proteins

Resuspend pellet in 1volume of B buffer supplemented by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M, anda dd 2/3 of volume of C buffer supplemented too by 10µl PIC 100X, 4µl PMSF 100mM and 4,15µl DTT 0,72M

Roll them at 4° for 45’

Pellet at max speed (at least 20000g) for 45’ at 4°

Supernatant contains nuclear proteins

Store at -80°

Proteins are ready do use.


Hypotonic A Bufer Pour 50ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 10mM 167µl
Tris pH 7.9 (1M) 10mM 500µl



Hypotonic B Bufer Pour 50ml
Glycérol 20% 10ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 10mM 167µl
Tris pH 7.9 (1M) 20mM 1000µl


Hypertonic C Bufer Pour 50ml
Glycérol 20% 10ml
MgCl2 (1M) 1,5mM 75µl
KCl (3M) 1,2M 20ml
Tris pH 7.9 (1M) 20mM 1000µl


i sent you by pm the protocols
fred

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finally i've decided to post these two protocols.

enjoy

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Thanks for sharing these.

Regards,
tltan

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can I replace NP40 with Triton-100x? Thanks

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for sure

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I need a protocol for transmembrane protein extraction from cells....do you have any protocol for this?
Thanks.

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