Protocol for preparation of the stock solutions

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
     - Common sense
     - Protocols
       - Stock solutions
       - Cell disruption
       - Protamin sulphate treatment
       - Analytical AM cut
       - Preparative AM cut
       - Protein precipitation by AM
       - Protein recovery from AM
       - Solubility of expression
       - Solubility of expression
       - Protein concentration
       - Protein concentration
     - 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.

Basic principles

Take a 1 litre glass beaker with a big magnetic follower bar and pour in the weight of salt powder that is required.
Pour miliQ water into the beaker to approximately the 900-950ml mark. Place on a stirrer and switch on. At the start you should help the magnet to start rotating by stirring the slurry with a rod or a spatula.
When the salt has dissolved pour the solution into a 1 litre volumetric flask. To make sure all the salt has been washed into the flask rinse the beaker with 2-3 small volumes of miliQ water and add them to the flask. Make the volume up to the 1 litre mark with additional milliQ water. You may have a problem with dissolving all the ammonium sulphate because 4M is close to the saturation point. To encourage dissolving, you can gently heat the mixture up to 40-50°C under constant control (to prevent overheating). Alternatively you can wait until most of the salt is dissolved, switch the stirrer off and gently pour the clear solution in to the volumetric flask. Add a little bit of milliQ water to the rest of the salt in the beaker, stir it briefly and add any clear solution to the volumetric flask. Do this 2-3 times until all the salt has dissolved.
Be careful and do not exceed 1 litre!!!
Filtrate solution through a 0.22µ filter using Filter Holder with Cellulose Nitrate Whatman filter or Stericup Filter Unit connected to a vacuum pump. Please note that 4M ammonium sulphate can not be filtered using the latter unit. Wet the membrane with a few drops of milliQ water before you start filtration.
Using the vacuum pump wash Filter Holder's porous disk with plenty of grey tap water immediately after use to prevent salt crystallising in the pores and therefore blocking the filter.
Pour solution into a clean bottle. Write the date and your initials on it. For a control measure, check the refraction of the solution. For 4M (NH₄)₂SO₄, it should be 37% and for 5M NaCl it should be 28% (on a sugar refractometer).

Preparation of 1M Tris-HCl pH 8.0

To prepare above solution you need to adjust the pH to 8.0 with concentrated HCl. The problem here is that tris buffer is temperature sensitive. Raising the temperature three degrees will make pH fall by 0.1. A lot of heat is produced during titration of 1M Tris solution with the concentrated acid so to make reproducible stock solution you need to titrate it on a water-ice bath with a thermometer placed in the solution. The standard temperature is 20°C, so you should make pH 8.0 at 20°C. Titration should be performed before you adjust volume. Because a significant volume of concentrated HCl is required to adjust pH, tris powder should be dissolved in 60-70% of the final volume of solution.

Please note that dilution also leads to a decrease in tris buffer pH. Normally we use 50 mM Tris-HCl solution for purification (made from the above stock) and the actual pH at 20°C is about 7.7. However if we use it at 4°C the pH is at about 8.