Hi,

50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM PMSF, ratios of cell wet weight to buffer volume of 1:1 to 1:4, cool the cell suspension, additionaly you can add lysozyme, final concentration of 0.2 mM and incubate on ice for 30 minutes before sonication. 

Jarek

attached!

i m using pottasium phosphate buffer 100mM ; 2mM dithiothreotal

Hi there,

There is no golden recipe. What you need basically is a system in which your protein of interest is happy : buffer system at physiological pH (usually phosphate or Tris-HCl), salt for  ionic strength (usually NaCl or KCl), DTT or BME for reduction and protease inhibitor cocktail to avoid protein degradation. As you mentioned Histag, avoid EDTA in the buffer and maintain reductant at low concentration to avoid precipitation on NiNTA.The important things are also the density of cells in the lysis buffer (I resuspend cells in around 1/20th of the original culture volume) and the temperature (keep it as cold as possible during sonication cycles).

https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/

I use 50 mM sodium phophate pH 7.5, 300 mM NaCl, 20 mM imidazole and 5 mM beta-ME. 30-50 mL lysis buffer for each liter of bacterial culture. 

Do you need to make it fresh? --- can I store it and if so how

Hi Adam Radek Martinez

It is better to make it fresh.

But, I usually make 10x Buffer containing Tris-HCl, NaCl, and EDTA with 10x final concentration that I will use later. And store it at 4°C.

So at the day I want to use, I will add the rest of the ingredients (DTT, protease inhibitors, etc).