Proteomics Facility


Protocol

In-gel Tryptic Digestion Protein Digestion Related Links

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

OR

- 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

Iodoacetamide solutions.

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.


Return to Top
Protein Digestion on PVDF Membrane
Protein bands have to be visible by Coomassie-blue staining or even better by Ponceau-S staining:

1) Cut out bands of interest, remove completely the areas without protein.

2) If membrane is dry, wet with little methanol.

3) Destain blots completely, if possible. Destain with acetone if stained with Coomassie or with water or 0.1% acetic acid 5% ACN if stained with Ponceau-S (10-15 min). The complete removal of especially Coomassie is important since it inhibits trypsin. Then rinse with water one time.

4) Incubate blots with 200 ml of 0.25% PVP-360 in 0.1% acetic acid for 20 min.

5) Rinse the blots with copious amounts of water, 6-10 times.

6) Dry bands in open air.

7) Cut the bands into small pieces using razor blade or scissors and place them back into the same tube.

8) Add 50 ml of digestion buffer: 5ml CH3CN, 45 ml 100 mM ammonium bicarbonate, pH 8.0. Check pH at this point with small piece of pH paper.

9) Add trypsin in the ratio of 1:10, mole/mole protein, at 37oC overnight.

10) Subject sample to MALDI analysis.

Protocol developed by Melanie Lin (Perseptive Biosystems), modified by Maria Person (UT-Austin).

Return to Top
Related Links
Protein digest protocols:

In-gel digest
Membrane digest
Prowl Recipes

Database Search Engines:
Prospector
Matrixscience
Prowl

Databases:

UniProt
NCBI
OWL

Return to Top


(Download PDF Reader.)

Last Reviewed: August 10, 2011


Maria D. Person
Director

Michelle V. Gadush
Manager

Andre Bui
Technician

John P. O'Brien
Post-doctoral researcher

Delivery Address:

Proteomics Facility
The University of Texas
at Austin
2500 Speedway MBB 1.420
Stop A4800
Austin, TX, USA
78712-1639

Email Address: pmaf
@austin.utexas.edu

Phone:
(512)471-2895

Institute for Cellular and Molecular Biology (ICMB)

index of the major headings: a b c d e f g h i j k l m n o p q r s t u v w x y z