Coomassie Stained SDS-PAGE In-gel Trypsin Digestion Protocol

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Coomassie stained SDS-PAGE In-gel Trypsin Digestion Protocol

Required solutions

All M.S. or HPLC grade

• HPLC grade H2O [1]
• 100 mM Ammonium bicarbonate (Ab)[2]
• 100% Acetonitrile (ACN) [3]
• 100 mM Ab : ACN = 50 : 50 (v:v)
• 250 mM DTT [4] in 100 mM Ab
• 50 mM Iodoacetamide (IAA) [5] in 100 mM Ab
• Trypsin/Lys-C Protease Mix [6]in powder
• 50% ACN, 5% Formic acid [7]
• 0.5% TFA in 5% ACN

Part I. Prepare gel band

1. Run regular SDS gel as needed;
2. Coomassie stain 30 min;
3. De-stain several times until gel bands are clear to see;
4. Keep gel in HPLC grade H2O O/N, gently shake;
5. Wash whole gel piece with HPLC grade H2O 2 x 30min, shake gently;
6. Cut desired gel band by new razor. Reference protein marker to determine protein size. Gel bands should be cut into 1 mm3. Smaller the better.
7. Put all pieces of one band into a 2 mL microcentrifuge tube. 2 mL has flatter bottom, which makes gel pieces sink into solutions easier.

Part II. Pre-digestion process

1. Wash cycle:
   1. Add enough volume of 100 mM Ab to cover all gel pieces, shake at 600 rpm, 37°C, 15 min. Then discard solution.
   2. Add enough volume of 100 mM Ab : ACN = 50 : 50 (v:v) to cover all gel pieces, shake at 600 rpm, 37°C, 15 min, or until gels are colorless by repeat a) and b) cycle. Then discard solution.
   3. Add enough volume of 100% ACN to cover all gel pieces, shake at 600 rpm, 37°C, 15 min. Then discard solution. Try to take out all visible solution, or speed vac to dry.

1. Reduction:
   1. Add enough volume of 250 mM DTT in 100 mM Ab to cover all gel pieces, shake at 550 rpm, 56°C, 1 hour. Discard solution.

1. Alkylation:
   1. Add enough volume of 50 mM IAA in 100 mM Ab to cover all gel pieces, incubate at room temp, 45 min. No shake. Cover by foil. Discard solution.

1. Perform all wash cycle as step 8 described.

Part III. Trypsin digestion

1. Prepare trypsin in 100 mM Ab. About 2 ug/band;
2. On ice: add trypsin solution to enough cover gel pieces, then let sit on ice for at least 30 min;
3. Put samples in 37°C, shake O/N, 600 rpm.

Part IV. Peptide extraction

1. Centrifuge samples until all condensations on the top and side are went down;
2. Transfer solution into a clean 1.5 mL microcentrifuge tube for every sample;
3. Add enough volume of 50% ACN, 5% FA to cover all gel pieces, shake at 550 rpm, 25°C, 20 min. Transfer solution into the 1.5 mL microcentrifuge tube in step 16.
4. Repeat step 17 one time.

Part V. Peptide clean-up

1. Dry samples from step 18 by speed vac;
2. Resuspend samples in buffer A, see Mobile Phases
3. Measure peptide concentration; (Dilute sample to save peptide if needed)
4. Depends on peptide quantity, combine or divide samples, then clean-up by C18 spin column.