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Freeze/Thaw procedure in cell lysis - How is this done??? (Jul/10/2006 )

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Hello,
I need to do do a western blot in MCF7 cells, and I am confused about how to do the cell protein extracts. I will use to lysate RIPA buffer and the freeze/thaw procedure, but I dont know the order in which i have to do this. I have this protocol, im not sure if its right. Please help me. sad.gif
1. wash cells in cold PBS and remove the PBS
2. add 10ml of iced cold PBS and scrape the cells , pellet the cells and resuspend in RIPA
3. aliquot and incubate on ice for 30min
4. sonicate <------ I can not sonicate, I need to freeze/thaw, but I dont know where in this steps should it go, and how to do it (should I freeze/thaw in RIPA buffer? or PBS?? )
5.centrifuge , remove supernatant and store @ -80

Please, could anybody tell me how to do this??

Thank you

-medchemgirl-

Freeze and thaw 3X in any buffer is sufficient to break cells.

What is the compostion of RIPA? RIPA may be better choice if contains protease inhibitors.

Unless your particular protocol requires this as a treatment step, you dont need to keep cell suspension in RIPA for 30 min on ice. you can go directly to F-T 3X.

-genehunter-1-

You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein...

-swanny-

Yes, you add protease inhibitors to RIPA:
* 150 mM NaCl
* 50 mM Tris, pH 7.4
* 1 % NP-40
* 0.25 % Sodium deoxycholate
* EDTA 1 mM
when I look for RIPA recipe they are all different, some use 50mM HEPES instead of Tris, is there a difference in this?

Thanks a lot, your suggestion clarified my mind. smile.gif

QUOTE (genehunter-1 @ Jul 11 2006, 12:39 AM)
Freeze and thaw 3X in any buffer is sufficient to break cells.

What is the compostion of RIPA? RIPA may be better choice if contains protease inhibitors.

Unless your particular protocol requires this as a treatment step, you dont need to keep cell suspension in RIPA for 30 min on ice. you can go directly to F-T 3X.

-medchemgirl-

Your mixture already has very strong membrane lytic activity, it should be sufficient to lyse cells in 5-10 min on ice and F-T is not needed. PBS does not contain detergents, such as NP-40 and SDS, so FT 3X will be needed.

Buffer tris and hepes makes little difference, other than that tris has a weak amine and may react with amine-reactive agents, such as glutaldehyde. You dont have to worry about it for your cell lysis experiment.

I suggest that you add protease cooktail to reduce protein degradation.

If you are studying cytosolic proteins, this will be enough for sample prep.

-genehunter-1-

If you don't have a sonicator, you can also pass your extracts through the needle of a siringe. You should use the 1ml siringe with the very small needle (like the one people use for insulin or drugs)

-dnafactory-

QUOTE (swanny @ Jul 11 2006, 08:52 AM)
You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein...


as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

-kylvalda-

This is great!! thank you very much for the suggestions.

I feel so happy I count with the support and advise of all of you!! rolleyes.gif

QUOTE (genehunter-1 @ Jul 11 2006, 02:54 AM)
Your mixture already has very strong membrane lytic activity, it should be sufficient to lyse cells in 5-10 min on ice and F-T is not needed. PBS does not contain detergents, such as NP-40 and SDS, so FT 3X will be needed.

Buffer tris and hepes makes little difference, other than that tris has a weak amine and may react with amine-reactive agents, such as glutaldehyde. You dont have to worry about it for your cell lysis experiment.

I suggest that you add protease cooktail to reduce protein degradation.

If you are studying cytosolic proteins, this will be enough for sample prep.

-medchemgirl-

I am making RIPA from scratch, I won't have manufacturer instructions, but I dont think I will be adding lysozyme and DNase.


QUOTE (kylvalda @ Jul 11 2006, 03:27 AM)
QUOTE (swanny @ Jul 11 2006, 08:52 AM)

You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein...


as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

-medchemgirl-

QUOTE (medchemgirl @ Jul 11 2006, 09:06 PM)
I am making RIPA from scratch, I won't have manufacturer instructions, but I dont think I will be adding lysozyme and DNase.


QUOTE (kylvalda @ Jul 11 2006, 03:27 AM)

QUOTE (swanny @ Jul 11 2006, 08:52 AM)

You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein...


as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity



I tried adding DNase though manufacturer said it wouldn't work and it made no difference on the gl so just leave it be... wink.gif

-kylvalda-

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