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-   -   Osmotic shock - solvent vs culture volume? (http://www.molecularstation.com/forum/protein-forum/26674-osmotic-shock-solvent-vs-culture-volume.html)

Julio 03-19-2006 03:20 PM

Osmotic shock - solvent vs culture volume?
 
Hi everyone.

Have a question about standard procedure of osmotic shock. I grow my
E.coli after induction by means of IPTG for 12-16h in 27Cdeg. Than I
want to make osmotic shock. Many people say many things about
proportions of Tris, sucrose, EDTA solution vs volume of culture after
harvesting by cetrifugation. For me the lower volumes the better
situation for next steps (eg batch with Ni-NTA resign). I read about
80mL of Tris, sucrose, EDTA solution for 1g of culture after spin but
also 8mL of this solution for 100mL of culture volume befor spin. What
do you advise me?

JC


DK 03-20-2006 04:13 AM

Osmotic shock - solvent vs culture volume?
 
In article <1142781643.925110.315030@z34g2000cwc.googlegroups .com>, "Julio" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

If this is for protein purification and you intend to do sonication
anyway, the osmotic shock adds little on top of that.
Without sonication, osmotic shock still won't do you much
without lysozyme and EDTA (don't forged to add ~ 2X Mg2+
over EDTA back after lysis before doing IMAC)

My advise is to add lysozyme *and* sonication.
In either case, I use ~ 10 ml of lysis solution/1 g of wet cells.

DK

Julio 03-20-2006 07:08 AM

Osmotic shock - solvent vs culture volume?
 
Dear DK

How much lysosyme u use. I suppose u add it to lysis solution. Next
question is about combination of sonication and osmotic shock. Should I
sonicate the cells when they are in lysis buffer? I used do use
sonication in ultrasound batch, however many proteins appeared I I had
problem with purification. However if it is possible to sonicate the
cells in lysis buffer or in the presence of Mg++ it would be really
graet for my yiels I suppose,

JC


DK 03-20-2006 07:32 AM

Osmotic shock - solvent vs culture volume?
 
In article <1142838492.855062.115710@i40g2000cwc.googlegroups .com>, "Julio" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Below is what I do but it's VERY flexible. Use common sense.

Lysis is 20 mM tris 8.0, 1 mM EDTA, 0.1 mg/ml lysozyme.
~ 10 ml/1 g wet cells. Let sit in the cold room in glass or stainless
steel beaker for ~ 30 min in the presence of ~ 1 mM PMSF.
Then add protease inhibitors cocktail and sonicate very thorouhgly
with intermittent mixing keeping in ice/water bath.

I use "turkey" style termometer right in the beaker and never
allow temperature to rise above 8-10C. Spin 15-20K, take sup,
add NaCl from 4M stock to ~ 300 mM, add MgCl2 to ~ 2 mM,
add imidazole according to what is best for your protein, spin
again - in ultracentifuge at 40-70K if available.

DK




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