Large Scale Digestion

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Large scale in-solution digests (for 2D-LC analysis)

Reagents:

1. 4X digestion buffer: 8 M electrophoresis grade urea (deinionized), 1.0 M Tris, 0.2 M methylamine, 8 mM CaCl2 (pH 8.5) (freeze in 1 ml aliquots).

2. 0.9 M Dithiothreitol (DTT) solution (35 mg into 0.25 ml of water) Make up fresh.

3. 1.0 M Iodoacetamide (IAA) solution (46 mg into 0.25 ml). Make up fresh.

4. Dissolve 100 g vial of trypsin gold (ProMega) in 100 l of water, keep on ice.

Protocol:

1. Start with a vacuum concentrated sample containing 1-5 mg of protein that contains nothing that would inhibit the digestion (protease inhibitors, detergent, buffers with a low pH). The volumes given below assume a 1 mg portion. Scale up if more than 1 mg is used.

2. Dissolve the protein sample by adding 100 L of 4X digestion buffer.

3. Add 12.5 L of the DTT solution, vortex, spin down to the bottom of the centrifuge tube and incubate at 50˚C in the thermocycler or water bath for 15 min.

4. Remove the sample from the thermocycler, let it cool for several min, add 25 L of the IAA solution, and incubate in the dark at room temp for 15 min.

5. Add an additional 12.5 L of DTT solution to the mixture.

6. Add 210 L of water. Remove 10 L for analysis by SDS-PAGE to determine the extent of digestion.

7. After 30 min at room temp check pH by spotting 1 L onto pH paper. If not pH 8.5 adjust by slow addition of 1 N NaOH.

8. Add 40 L of 1 g/L trypsin gold (1:25 ratio of enzyme to substrate), vortex gently to mix, centrifuge to the bottom of the tube and incubate overnight at 37ºC.

9. Remove 10 L for analysis by SDS-PAGE to determine the extent of digestion.

10. Add 20 L of neat 88% formic acid to stop the digestion.

11. Sep-Pak clean the sample to remove all salts prior to loading on the polysulfoethyl A column.