His-tag protein purification- buffers to use - What buffers to choose for His-tag chromatography (Jan/28/2008 )

Hi all! I am going to express and purify a His-tagged protein in E.coli in the next few weeks. Since this is the first time I am going to do this, I have a few questions:
1. What are the buffers I should use? I will need a buffer for resuspending my cells, one for equilibrating my column, one for washing the column and one for eluting, and then one more for storing the protein in. How should I choose the composition of all these buffers? What is the rough salt concentration to be used for each step?

2. Is Glycerol necessary in the buffers for protein?

3. Some protocols recommend phosphate based buffers, others use Tris-based or Hepes based buffers- what is the difference between them and how to choose one over the other?

I will be grateful for any help from experienced people about this.

Thank you.

There is a lot of information regarding His-tag protein purification systems, its depends which company u buy from.

i use qiagen protocols.

i upload the file for u.

use it wisely

If you are using Qiagen's nickel agarose beads, there is a lot of information in the manual there. The difference in buffers seem to dependent on what you are doing with your protein. I have used Tris for my normal proteins, used in certain in vitro assays. I use HEPES based buffers for buffers being added into egg extracts (Tris is bad for this system).
What kind of column are you using? I don't have a separate equilibration buffer because I don't need it.. the beads have already been washed and that is what is being loaded into the column. (ie, its not like a silica based column for DNA preps)
I only add glycerol to dialysis buffer for certain proteins that tend to precipitate, and my buffers use 500mM salt.
It's also helpful to look at the experimental section of a paper, if you're making this protein to replicate a published assay.