In-gel alkylation and digestion with modified Trypsin

1. Excise the stained protein band from the gel with a scalpel. Cut the gel slice into ca. 11 mm cubes, and put the pieces in an Eppendorf tube.

 

2. Remove Coomassie stain by rinsing the gel pieces twice in 200 microl 0.2 M NH4HCO3 (Ambic; 0.1 Mfinal conc.)/Acetonitril (ACN)1:1, for 45 min at 37C.

 

3. Shrink the gel pieces by adding 100 microl ACN at room temperature (wait until the pieces become white), and then remove ACN. Dry in vacuum centrifuge for 5 min.

 

4. Rehydrate the gel pieces in 20 mM DTT in 0.1 M Ambic ( 50 microl) for 30 min at 56C.

 

5. Remove excess liquid (but do not dry in vacuum), and shrink the pieces with ACN as above (step 3). Remove ACN, and add 55 mM iodoacetamide in 0.1 M Ambic ( 50 microl) for 15 min at room temperature in the dark.

 

6. Remove excess liquid, and wash the pieces once with 0.1 M Ambic. Remove Ambic and shrink the pieces with ACN. Remove ACN, wash with H2O and shrink again with ACN. Remove ACN and dry in vacuum centrifuge for 5 min.

 

7. Add 10 microl of 0.05 microg/microl Promega’s modified trypsin solution in 0.1 M Ambic and 10 ACN, e.g. 80 microl Ambic, 10 microl ACN and 10 microl of 0.5 microg/microl trypsin solution. Allow to absorb for 10 min at room temperature. Then add solution of 0.1 M Tris-HCl, pH 9.2 and 10 ACN to completely cover the gel pieces. Incubate overnight at 37C in the dark.

 

8. Collect the liquid into Eppendorf tube. Extract the peptides from the gel pieces twice with 150 microl of 0.1 trifluoroacetic acid (TFA)/60 ACN (in water) for 30 min at 37C. Collect the liquid in the same eppendorf tube as previously. Dry down the collected solution to 30 microl in vacuum centrifuge, then add100 microl 0.1 TFA and again dry down to 30 microl. The prep is now ready for HPLC separation or desalting in the microtip.

  • 20 mM DTT 3.08 mg DTT/1 ml 0.1 M Ambic;

  • 55 mM iodoacetamide 10.2 mg/1 ml 0.1 M Ambic;

  • Seq. grade modified trypsin, porcine, sp.act. 500 IU/mg; Promega, Madison, USA, V511A 11652007 (20 microg of trypsin in 40 microl of buffer: 0.5 microg/microl)

  • Acetonitril; Rathburn Chemicals Ltd., Walkerburn, Scotland; No. RH 1015, HLPC grade;

  • Ammonium bicarbonate (Ambic); Sigma, A-6141, Fw 79.06 g/mol;

  • Trifluoroacetic acid; Sigma, T-6508, Fw 114.0 g/mol.

 

2002 06 20 – Ove Julius