Stock solutions preparation

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
       - Equipment & Reagents
       - Stock solutions
       - Chromatographic columns
       - Crude extract
       - pre Chromatographic step
       - Chromatographic step
     - Common sense
     - Protocols
     - Charts & Tables
     - Appendix
   - Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

Stock solutions preparation

The most useful stock solutions in protein purification are:

5M NaCl; 4M (NH₄)₂SO₄; 1M Tris-HCl, pH 8.0.

To prepare 1 litre of solution you need:

For 5M NaCl	292 g
For 4M (NH₄)₂SO₄	528 g
For 1M Tris-HCl	121 g

Other stock solutions:

You may need EDTA stock solution. Due to low solubility of this compound concentration of stock solution is 0.2M - 0.25M. You should adjust pH of the solution to 6.5 - 7 with 5M NaOH before you adjust it to required volume.

You may also need:
1M HEPES-NaOH pH 7.0 or 7.5
1M MES-NaOH pH 6.0 or 6.5

Those buffers belong to Good's buffers and they are not temperature sensitive so there is no need to control temperature during their preparation, opposite to tris-buffer (see Protocols section for details)

It is best to keep stocks of buffers in the fridge.

The sequence of procedure is as follow:

Weighing powder -> dissolving it in water -> titration (for buffers and EDTA only) -> adjusting volume -> filtration (see Protocols section for details)

By having stock solutions you can prepare any buffer for protein purification in seconds. Simply pour required volumes of stock solutions in to a Duran bottle and adjust the volume with ultra pure water to the top mark on the bottle.

Example: To prepare buffer 50mM tris pH 8, 100 mM NaCl, 2mM EDTA; pour 50 ml of 1M tris, 20 ml of 5M NaCl and 10 ml of 0.2M EDTA in to a 1 litre bottle and add ultra pure water to 1 litre mark.