In-gel alkylation and digestion with endoproteinase Glu-C (protease V8)

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 Glu-C solution (in 25 mM Ambic pH 7.8 and 5 ACN). Allow to absorb for 10 min at room temperature. Then add solution of 25 mM Ambic, pH 7.8 and 5 ACN to completely cover the gel pieces. Incubate overnight at 25 or 37C in the dark (see the comments!).

 

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 25 or 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 endoproteanase Glu-C, Staphylococcus aureus V8; Roche (Boehringer Mannheim), Germany, 1047817 (50 microg of Glu-C in 100 microl of water: 0.5 microg/microl) or from Sigma, P6181.

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

  • Ammonium bicarbonate (NH4HCO3) (Ambic); Sigma, A-6141, Fw 79.06 g/mol; 25mM solution: 197.65 mg/100 ml water, pH is 7.8 without any adjustment;

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

 

NB! For peptide or protein digestion, a ratio between 1/20 and 1/100 (w/w) of enzyme to substrate is recommended. ’Roche’ suggests to use 25 mM ammonium carbonate buffer, pH 7.8 and in order to avoid autolysis the incubation temperature should not exceed 25C! While according to ’Sigma’ the buffer has to be 100 mM of ammonium carbonate, pH 7.8 and the incubation temperature 37C!

 

2003 06 20 – Julius