In Gel Digestion

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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.