Protocol for analytical ammonium sulphate cut (AM cut)

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1. Take 0.5 ml of the cold crude extract and add 0.3 ml of 4M ammonium sulphate solution (this gives 1.5M of ammonium sulphate). Spin down for 2 min in the bench centrifuge or, better still, in a refrigerated centrifuge. Place supernatant fraction into a fresh tube. Re-suspend pellet in 0.1 ml of buffer A (50mM tris-HCl, pH 8.0).
   Mark this tube: 1.5M pellet.
2. To the supernatant fraction add 0.2 ml of 4M ammonium sulphate, repeat centrifugation. Again decant supernatant fraction in to a fresh tube. Keep on ice. Dissolve pellets in 0.1 ml of buffer A. Mark this tube: 2.0M pellet.
3. Weigh out 66 mg of ammonium sulphate and dissolve in the supernatant fraction. Repeat procedure as above. Keep on ice or at 4°C. Mark this tube: 2.5M pellet.
4. Repeat step 3 two more times to have 3M and 3.5M pellets and 3.5M supernatant.
5. Check protein concentration in each tube. Most probably you will find a very low protein concentration in the 3.5M supernatant fraction.
6. Run gel to see which fractions contain the target protein. Take equal volumes from all fractions to prepare samples for the gel. You may have a problem with 3.5M supernatant fraction as it will have a very high salt concentration and low protein concentration. If you recover less than 10% of total protein from this fraction it is best to exclude it from the analysis. If there is a significant amount of protein, dilute it 4 fold with buffer A and concentrate to 0.1 ml using VivaSpin6 concentrator.
7. Analyse the pattern on the gel and develop preparative ammonium sulphate cut strategy.