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.

Solution Preparation:

8M Urea (480mg/ml) in 50 mM AMBIC  (ammonium bicarbonate)  (During digestion ~2M)

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

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

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

10% TFA in water


  1. Add 15 uL of 8M Urea in AMBIC to the dried sample to denature protein.
  2. Add 2 uL of 50mM DTT stock to reduce disulfide bridges.
  3. Incubate for 35 min at 45 oC.
  4. Cool to room temperature. Add 5 uL 50mM iodoacetamide stock to alkylate the free cysteines.
  5. Incubate for 30 min at room temperature in the dark.
  6. Quench unreacted iodoacetamide by adding 5 uL of 50 mM DTT and incubating for 15 min at room temperature in the dark.
  7. Add 2.5 uL 25 mM CaCl2 and 22.5 uL * 50 mM AMBIC, to improve trypsin activity.
  8. Make sure that urea concentration is < 2 M before adding trypsin.
  9. 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.
  10. Incubate overnight at 37 oC.
  11. 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).
  12. Dry down the samples.
  13. Resuspend the peptides in 20 uL of 0.1% formic acid in water.
  14. Desalt using uC18 ZipTips.
  15. Dry down the samples in a speed vac.
  16. 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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