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Procedure for In-Gel Digestion 

 

 

Before proceeding, review the Contamination Issues document. 

Solution recipes you will need are in the Recipes document. 

Necessary resources for the digestion protocol are here.  

A-Excise and destain protein bands/spots 

⦁ Rinse the one- or two-dimensional gel with water for 10 min  
⦁ Place the gel mounted on a clean and wet glass plate onto a light box and excise bands (spots) of interest with a clean scalpel. 
 ⦁ If necessary, cut excised bands (spots) into smaller pieces (ca. 1 × 1 mm). Note: Be careful when using a pipette suction to move spots since smaller pieces could clog the pipette tip. 
 ⦁ Transfer gel pieces into a microcentrifuge tube. Add enough destain solution to cover. 
 ⦁ Spin them down and let sit for 10 min. 
 ⦁ Carefully remove and discard the destain solution and repeat the process until gel pieces become transparent. 
 ⦁ Remove the last destain solution and add ACN to dehydrate the gel pieces and let sit for 10 min, shaking every couple of minutes. 
 ⦁ Repeat the procedure (2 or 3x) until the pieces become completely dry (white).  
PAUSE POINT: Store samples in desiccator at RT for 1 day or -20°C up to 1 month.  

 
 
 

B- Reduction and Alkylation 

⦁ Add 20 µL of 20 mM DTT solution and incubate 45 mins at 56°C 
 ⦁ Allow tubes to cool to RT, then add 20 µL of IAA solution and incubate in the dark for 40 mins. 
 ⦁ Carefully remove all solutions and add ACN to dehydrate the gel pieces and let sit for 10 min., mixing during this time. 
 ⦁ Repeat the procedure (2 or 3x) until pieces becomes completely dry (white).  

 
 
 

C- In-gel Digestion 

⦁ Rehydrate the gel pieces with 20 µL of the trypsin solution (or enough to cover the dry gel pieces) per tube and let sit 20-30 mins. 
 ⦁ After ca. 30 min, check if all the solution was absorbed and add more trypsin buffer, if necessary. 
 ⦁ Leave the gel pieces for another 30 min. then add 10–20 μl of 50 mM AmBic to cover the gel pieces and keep them wet during enzymatic cleavage. 
 ⦁ Place the tube in a pre-heated water bath at 37°C and incubate overnight  

D- Peptides Extraction 

⦁ Remove tubes from water bath and spin down gel pieces using a microcentrifuge. 
 ⦁ Add 15 µL of Stop solution to each sample to stop the enzymatic reaction. 
 ⦁ Collect the supernatant in a new labeled tube. 
 ⦁ Add 50 µl of Elution solution A (or enough to cover) and incubate at 40°C for 15 min., vortexing every 3 min. (or in a thermomixer). 
 ⦁ Collect the supernatant and add to the tube from step 3. 
 ⦁ Add 50 µl of Elution solution B (or enough to cover) and incubate at 40°C for 15 min., vortexing every 3 min. (or in a thermomixer). 
 ⦁ Collect the supernatant and add to the tube from step 3. 
 ⦁ Add 50 µl of ACN (or enough to cover) and let sit for 15 min., vortexing every 5 min. 
 ⦁ Collect the supernatant and add to the tube from step 3. 
 ⦁ Repeat steps 8 and 9. 
 PAUSE POINT: Dried extracts can be stored at -20°C for a few months.  

 
 
 

E- Peptides Desalt 

Samples: Before starting, resuspend samples in 10 µL of Equilibrium solution.  
 
Note: once you start, do not let the Ziptips dry 
  

Equilibrium:  

⦁ Aspirate 10 µL of wetting solution I into tip and dispense to waste. Repeat 7-10 times. 
 ⦁ Aspirate 10 µL of wetting solution II into tip and dispense to waste. Repeat 7-10 times. 
 ⦁ Aspirate 10 µL of equilibration solution into tip and dispense to waste. Repeat 7-10 times.  

 
 

Binding and wash  

⦁ Bind peptides to ZipTip by aspirating and dispensing 7-10 cycles. 
 ⦁ Aspirate washing solution and dispense to waste. Repeat wash twice.  

 
 

Elution:  

⦁ Aspirate 10 µL of elution solution into tip and dispense into a new tube. 
 ⦁ Aspirate and dispense eluant through ZipTip at least 3 times without introducing air. 
 ⦁ Dry down using speed vacuum. Dried extracts can be stored at -20°C for a few months.