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Trypsin In-Gel Sample Digestion Protocol
This protocol was developed for 2D gel spots, ~100ng, using Dilution A of trypsin for silver, sypro ruby or faint Coomassie stained bands. If Coomassie stained spots at ug levels (medium to dark) are being digested, use Dilution B for trypsin aliquots.
Be ultraclean in all steps prior Day 3 step 1. If any keratin from dust or skin gets into the sample from the gel, cutting surface, pipette tips, tubes or digestion buffer, it will show up in the mass spectra and swamp weaker signals from the protein. Use tubes and tips that have not been exposed to ungloved hands. If possible work in the hood or a clean bench and filter the solutions, keep all the materials in an area dedicated to in-gel digestion. Wear lab coat and keep hair covered and held back.
I. Solutions Preparation:
Note: Put all reagents in polypropylene tubes and use highest quality HPLC grade reagents.
1. Destain (400uL/sample)
- 50% Methanol, 5% Acetic Acid in HPLC water
2. Trypsin (20uL/sample)
- Add 160uL of Trypsin buffer to Trypsin stock solution tube (40uL of 0.5ug/uL). Then make aliquots of 20uL (2ug of Trypsin) and store in the freezer.
- Prepare a fresh solution from aliquot before use.
- Dilution A: 180uL of 50mM Ammonium Bicarbonate to each vial of 2ug (Promega) sequencing grade Trypsin. (Final concentration: 10ug/mL = 10ng/uL). 200 uL per vial
- Dilution B: 20uL of 50mM Ammonium Bicarbonate to each vial of 2ug (Promega) sequencing grade Trypsin. (Final concentration: 50ug/mL = 50ng/uL). 40 uL per vial
3. Acetonitrile (600uL/sample)
4. 10mM DTT (100uL/sample)
- Prepare a fresh solution before use.
- 1.54mg of DTT in 1mL of 100mM Ammonium Bicarbonate
5. 50mM Iodoacetamide (100uL/sample)
- Prepare a fresh solution before use.
- 10mg of Iodoacetamide in 1mL 100mM Ammonium Bicarbonate
6. 100mM Ammonium Bicarbonate (200uL/sample)
- 0.79g of Ammonium Bicarbonate in 100mL of HPLC Water (Filtered with 0.2um syringe filter)
Note: Also add amount needed to make 50mM Ammonium Bicarbonate, 10 mM DTT and 50mM
7. 50mM Ammonium Bicarbonate (20uL/sample)
- Dilution 1:1 of HPLC Water:100mM Ammonium Bicarbonate
Note: Also add amount needed to make Trypsin solution.
8. Extraction buffer 1 (20uL/sample)
- 5% Formic Acid in HPLC Water
9. Extraction buffer 2 (20uL/sample)
- 1:2 (v:v) 5% Formic Acid in HPLC Water : Acetonitrile
II. Sample digestion
A. Day 1 (steps in Day 1
1. Cut the spot and chop it with a clean razor blade into 1mm3 pieces. If Coomassie gel is used, cut spot as close as possible to minimize gel volume.
2. Pick the gel pieces with the razor or with a wide bore P1000 tip or P1000 tip with end cut off and put them in a 1.5mL microfuge tube
3. Add 400uL of Destain solution. Spots can be left in Destain solution indefinitely at 4oC.
B. Day 2 (microfuge briefly before each solution removal step)
1. Remove and discard Destain solution.
2. Add 200uL of Acetonitrile to dehydrate. Gel pieces should turn white. If they do not, repeat Acetonitrile treatment.
3. Remove and discard Acetonitrile solution.
4. Air dry at room temperature until gel pieces no longer stick to the tube or each other.
5. Add 100uL of 10mM DTT solution to reduce the gel.
6. Incubate for 30min at room temperature.
7. Remove and discard DTT solution.
8. Add 100uL 50mM of Iodoacetamide solution to alkylate the gel
9. Incubate for 30min at room temperature.
10. Remove and discard Iodoacetamide solution.
11. Wash with 200uL of 100mM Ammonium Bicarbonate solution.
12. Incubate for 10min at room temperature.
13. Remove and discard Ammonium Bicarbonate solution.
14. Wash two times with 200uL of Acetonitrile solution to dehydrate the gel. Incubate each time for 5min at room temperature. Gel should turn white.
15. Air dry at room temperature until gel pieces no longer stick to the tube or each other.
16. Prepare Trypsin solution (See solution preparation for recipe). Choose either Dilution A or B based on amount of protein in gel band.
17. Add 20uL of Trypsin solution to each spot (sample), keep cold until gel is rehydrated. If gel is not fully hydrated, add 20uL of Trypsin solution again.
18. Incubate in ice for a few minutes.
19. Add 20uL of 50mM Ammonium Bicarbonate solution to each spot (sample).
20. Incubate overnight at 37oC.
Delivery Address: Proteomics Facility Email Address:
Maria D. Person
Michelle V. Gadush
John P. O'Brien
The University of Texas
2500 Speedway MBB 1.420
Austin, TX, USA