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Lys-C OR Trypsin In-Solution Protein Digest Protocol

Original protocol from the  J. Yates Lab, Scripps Research Institute

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. Add in Lys-C 1µl (0.1µg/µl), 1/100th total amount.
  2. Incubate for 4 hr. at 37°C in the dark.
  3. Add formic acid to 5% final conc.



for 1ml:

1ml MilliQ water
make a 1/10 dilution and store at –20°C in aliquots
500mM iodoacetamide

for 0.5ml:

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 easier printing.