4 replies to this topic

[rimal]

Hi all,

I need a recipe of protein buffer lysis, I found lots of formula thru googling but I cant decide which one is the suitable ?

also I have buffer formulas containing 25mM Tris-Hcl, 10% glycerol, 50mM NaCl, 0.1CHAPS and I want to prepare it from a stock but I could'nt because I dont know the ratio between each substance so can I prepare it all together if so how can I adjust the Tris-hcl buffer before adding the substance or adjut it after?

Edited by rimal, 27 June 2009 - 04:44 PM.

[T C]

Hey,

I don't think I undertsnad yr question but hope this helps:

The lysis buffer that I use has 50 mM Tris, 10 % glycerol, and 150 mM NaCl. This is the standard buffer that I use for all proteins but add different things depending upon the protein that I purify. For instane add protease inhibitors if you think proteases is a problem, increase NaCl to upto 500 mM if you see non specific proteins co-purifying etc.

Also, the buffer that I make is made from individual stocks. So from a 1M tris stock, use N1V1=N2V2 to get the volume required.
Add everything and makeup the volume to the final volume that you need.

And use ice cold buffers.

Best,
TC

[rimal]

T C, on Jun 27 2009, 10:09 PM, said:

Hey,

I don't think I undertsnad yr question but hope this helps:

The lysis buffer that I use has 50 mM Tris, 10 % glycerol, and 150 mM NaCl. This is the standard buffer that I use for all proteins but add different things depending upon the protein that I purify. For instane add protease inhibitors if you think proteases is a problem, increase NaCl to upto 500 mM if you see non specific proteins co-purifying etc.

Also, the buffer that I make is made from individual stocks. So from a 1M tris stock, use N1V1=N2V2 to get the volume required.
Add everything and makeup the volume to the final volume that you need.

And use ice cold buffers.

Best,
TC

this exactly what I want in another simple explanation I am looking for a nondenaturing protein lysis buffer?

[T C]

This is it then
Just change the buffer according to yr requirement: 50 mM tris/100 mM phosphate etc.

Best,
TC

rimal, on Jun 28 2009, 02:42 PM, said:

this exactly what I want in another simple explanation I am looking for a nondenaturing protein lysis buffer?

[baienoix]

Hi,

I am working on sperm cells and I am looking for a procedure to remove DNA after I have lysed these cells. To lyse, I am using a buffer with 1% Triton (which is compatible with DNAseI activity) but also 50mM DTT. Does someone has a idea to remove the released DNA without using centrifugation (I want to keep all the proteins), sonication or syringe?

Thanks for your help.