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Trypsin Solution Digest Protocol (updated 8/12/2025)

Trypsin Solution Digest Protocol (updated 8/12/2025)

This protocol was developed for purified proteins.  For tissue samples and cell lysates, we recommend protein precipitation and BCA after lysis and prior to digestion.

 

Digestion using TCEP and CAM (Updated 8/12/2025)

Solution Preparation:

8M Urea (480mg/ml) in 50 mM ammonium bicarbonate (during digestion ~ 1 M)

100 mM TCEP in water

400 mM Chloroacetamide in water

50 mM ammonium bicarbonate (AMBIC)

25 mM CaCl2 (2.7mg/ml) (Final: ~1 mM during digestion)

10% TFA in water

NOTE: Dry down the samples before starting 

  1. Add 20 μL of 8M Urea in AMBIC to the dried sample to denature protein.

  2. Add 2 μL of 100 mM TCEP stock and incubate for 20 min at 25 o C to reduce disulfide bridges. 

  3. Add 2 μL of 400 mM chloroacetamide stock and incubate for 20 min at room temperature in the dark to alkylate the free cysteines. 

  4. Quench unreacted chloroacetamide by adding 2 μL of 100 mM TCEP stock and incubating for 5-10 min at room temperature.

  5. Add 6 μL of 25 mM CaCl2 to improve trypsin activity (optional).

  6. Add 128 μL of 50 mM AMBIC to lower urea concentration to 1 M.

    • NOTE: urea concentration must be ~ 1 M before adding trypsin.

  7. Calculate and add the amount of trypsin required to achieve a 1:50 to 1:20 ratio of trypsin mass: protein mass. Our trypsin aliquots are at 100 ng/μL.

  8. Incubate overnight at 37 o C.

  9. Allow the digest to cool to room temperature and stop the digestion by acidification with TFA. Add 10-20 μL of 10% TFA (pH should be ~2).

  10. Dry down the samples.

  11. Resuspend the peptides in 20 μL of 0.1% formic acid in water.

  12. Desalt using μC18 Zip Tips.

  13. Dry down the samples in a speed vac.

  14. Resuspend in 7 μL of 0.1% formic acid in water.



Digestion using DTT and IAM

Solution Preparation:

8M Urea (480mg/ml) in 50 mM ammonium bicarbonate (during digestion ~ 1 M)

50 mM DTT (7.5mg/ml) in 50 mM AMBIC (Final: 5 mM during reduction) 

50 mM Iodoacetamide (9 mg/ml) in 50 mM AMBIC (Final: 10 mM during alkylation)

25 mM CaCl2 (2.7mg/ml) in 50 mM ammonium bicarbonate (Final: ~1 mM during digestion)

10% TFA in water

NOTE: Dry down the samples before starting 

  1. Add 10 uL of 8M Urea in AMBIC to the dried sample to denature protein.

  2. Add 2 uL of 50 mM DTT stock and incubate for 35 min at 45 oC to reduce disulfide bridges.

  3. Cool to room temperature. Add 5 uL 50mM iodoacetamide stock to alkylate the free cysteines.

  4. Incubate for 30 min at room temperature in the dark.

  5. Quench unreacted iodoacetamide by adding 5 uL of 50 mM DTT and incubating for 15 min at room temperature in the dark.

  6. Add 2.5 uL 25 mM CaCl2 and 22.5 uL * 50 mM AMBIC, to improve trypsin activity.

  7. Make sure that urea concentration is < 1 M before adding trypsin.

  8. Calculate the amount of trypsin to achieve a 1:50 to 1:20 ratio of trypsin mass: protein mass, for example, 2-5 ug of trypsin for 100 ug of protein in a 10 uL volume (in 50 mM AMBIC). Add 10 uL trypsin solution to the sample.

  9. Incubate overnight at 37 oC.

  10. Allow the digest to cool to room temperature and stop the digestion by acidification with TFA. Add 5-10 uL of 10% TFA (pH should be ~2).

  11. Dry down the samples.

  12. Resuspend the peptides in 20 uL of 0.1% formic acid in water.

  13. Desalt using uC18 ZipTips.

  14. Dry down the samples in a speed vac.

  15. Resuspend in 7 uL of 0.1% formic acid in water.

  • the amount of 50mM AMBIC can be coordinated with the volume of trypsin added.

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