Applied Biosystems: In-Gel Digestion Protocol

 

for Coomassie & Silver Stained 1D & 2D Gels

 

 

 

For Silver Stained Gels

 

1.     Prepare 30 mM Potassium Ferricyanide and 100 mM Sodium Thiosulfate stock solution.

2.     Cut Stained gel band with a scalpel and place into 500 uL microfuge tubes.

3.     Make destain solution by mixing the stock solutions at 1:1 ratio immediately before using.

4.     Apply 100 mL destain solution to each tube and vortex 10 minutes or until brownish color disappeared.

5.     Remove supernatant and wash with deionized water three times.

6.     Dice the gel with a scalpel inside the microfuge tube into smaller pieces.

7.     Wash with 50% Acetonitrile/25 mM Ammonium Bicarbonate for 15 minutes each three times.

8.     Follow the regular in-gel digestion procedure.

 

 

Standard procedure for In-gel Digestion

 

1.     Before cutting the gel pieces wash the gel well with water to remove acetic acid, methanol, and SDS and buffer salts.

  1. Cut gel spots and take them in 500microL eppendorf tube.

3.     Add some (100-200 microL) 50%AcCN/50% 50mM Ammonium Bicarbonate, pH8 to it and crush the gel pieces using a pestle and leave for 15 minutes.

  1. Centrifuge and remove supernatant and add another aliquot of the above 50% AcCN/Ammn. Bicarb solution vortex and leave 15 min at room T.
  2. Centrifuge. Repeat above step.  All the dye should be gone now.
  3. To the left over gel pieces add 100 microL AcCN and vortex and speed vac.

Reduction and Alkylation of cysteine residues (carbmidomethylation)

 

NOTE: If you have done this before running the gel than proceed to digestion directly.

 

1.     Add enough 5mM DTT solution (in ammonium bicarbonate, 50mM pH8) to the gel pieces to soak the gel pieces and incubate at 37º C for 30mins. 

  1. Wash with 50% acetonitrile in ammonium bicarbonate with 100microL twice. 
  2. Add 100microL acetonitrile and vortex and speedvac to dryness. 
  3. Add 10mM Iodoacetamide solution to the gel pieces (enough to soak the gel pieces) and incubate for 30mins at room temperature. 
  4. Wash twice as above and dry the gel pieces in acetonitrile and proceed for digestion step.

 

 

Trypsin Digestion

 

If the band is dark, with coommassie stain add stock trypsin to the gel slices so that the gel slices are just covered with a thin layer of the solution.  Use Promega Trypsin in 50mM Ammonium Bicarbonate pH8-8.5 (stock solution - 20micrograms in 2mL buffer.) 

 

Note: If too much trypsin solution is added there will be to many trypsin peaks in your mass spectra. To avoid this you may want you add ammonium bicarbonate buffer.

 

If the gel slices are light, then dilute the stock trypsin so that each gel band gets about 2-4 microL of the stock trypsin. 

 

If the gel band is faint, (can hardly be seen), than lower the trypsin even further.  I go according to the calculation of protein to trypsin ratio of about 1:20 and a faintly stained coommasie band is about 0.1 microgram protein or less. 

 

  1. Leave at 37ºC overnight.
  2. Add 50% AcCN/0.3%TFA in water to extract the peptides (Volume roughly the same amount of gel you have in there.)
  3. Leave for 15min and spin and take supernatant and save.
  4. Add another 100microL of the above AcCN solution and leave for 15min spin and extract supernatant and save (In the same tube as above)
  5. If you want you can extract for the third time.
  6. Put the mixed supernatants in a speedvac and then spot and run on MALDI. The speedvac step can be repeated to chase the ammonium bicarbonate away, just add water and then speedvac again and repeat the process three times.

 

Ordering information:

 

Pestle

Vendor          Fisher Scientific

Phone #        800 766 7000

Description    Kontes Pellet Pestle, 0.5mL

Catalog#       K749521-0590

 

DTT                                                                          IAA

Vendor          Sigma                                                   Sigma

Phone #        800 325 3010                                        800 325 3010

Description    DL-Dithiothreitol                                     Iodoacetamide

Catalog#       D-0632                                                 I-1149

 

 

The pestle listed fits only the 500microL eppendorf tube.  Before using the pestle clean them well with ddH2O otherwise you will see a lot of keratin peaks due to the dust on the pestles.  Note: Please wear gloves when handling the gel and washing the pestles and through out the digestion and extraction procedure.

 

Note: There are several other ways of reducing the cysteines and alkylating them.  For reduction another well known reagent is triphenylphosohine and for alkylation, vinyl pyridine (this mode of alkylation helps in the MALDI, in the sense that the modification helps cysteine containing peptides to fly well.)  The vinyl pyridine reagent induces pyridyl ethylation modification of the cysteines.  Care has to be taken to keep the vinyl pyridine reagent dry.  The DTT solution can be made up in stock and frozen over a period of time.  But the alkylating agents have to be made fresh every time the digestions are being done (both iodoacetamide and vinyl pyridine.)