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Original protocol from the J. Yates Lab, Scripps Research Institute; revised May ’09 by Lori

If you have not already done so, please read our sample preparation tips.

  1. Bring solution up to 8M Urea and 100mM Tris-HCl pH 8.5 (use 10 M urea and 1 M tris stocks; ideal final volume is about 80µl).
  2. Add 100 mM TCEP (a reducing agent) to a final concentration of 5 mM.. Incubate at room temp. for 20 min (2.5µl if the total volume was 80µl).
  3. Add 500mM iodoacetamide (make fresh daily) to a final concentratrion of 10 mM. Incubate at room temp. for 15 min. in the dark (covered with foil).
  4. Use one of the following enzymes (trypsin is recommeded for mudPIT):

Trypsin Digest:

    1. Dilute samples by a factor of four with 100mM Tris-HCl pH 8.5 (final urea conc. = 2M; if sample volume is a problem, dilute by only a factor of two to 4M urea)
    2. Add 100 mM CaCl2 to a final conc. of 1mM.
    3. Add in trypsin 1µl (0.5µg/µl) Promega sequencing grade trypsin is recommended.
    4. Incubate overnight at 37°C in the dark.

Lys-C Digest:

    1. 1. Add in Lys-C 1µl (0.1µg/µl), 1/100th total amount.
    2. Incubate for 4 hr. at 37°C in the dark.

5. Add formic acid to 5% final conc.



for 1ml:

  • 287mg
  • 1ml MilliQ water
  • make a 1/10 dilution and store at –20°C in aliquots

500mM iodoacetamide

for 0.5ml:

  • 46mg
  • 500µl ddH2O, make fresh

1M CaCl2

for 100ml:

  • 14.7g CaCl2•2H2O
  • ddH2O to 100ml
  • filter sterilize

You may also access this protocol via a PDF for easy printing.