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University of Wisconsin–Madison

Mass Spectrometry / Proteomics Facility

Buffers and Solutions for In Gel Digests

500mM Ammonium Bicarbonate [(NH4)HCO3]

  • (NH4)HCO3-1.6g
  • Millipore water-to 40ml final volume
  • Filter through high protein binding filter [Nitrocelulose or PTFE; 0.45um pore size)

25 mM (NH4)HCO3

  • 500mM (NH4)HCO3-2ml
  • Millipore water-to 40ml final volume
  • Check pH (should be between 8-8.5)

25mM (NH4)HCO3/50% Acetonitrile [ACN]

  • 500mM (NH4)HCO3-2ml
  • ACN (HPLC grade)-20ml
  • Millipore water-18ml
  • Make sure pH stays at 8-8.5

500mM Ditiothreitol [DTT] stock

  • DTT-77.1mg
  • 25mM (NH4)HCO3-to 1ml final volume
  • Store at -20°C in 50ul aliquots

550mM Iodoacetamide [IAA] stock

  • IAA-101.7mg
  • 25mM (NH4)HCO3-to 1ml final volume
  • Store at -20°C in 50ul aliquots

30mM Potassium Ferricyanide (III) [K3Fe(CN)6]

  • K3Fe9(CN)6-494mg
  • Millipore water-to 50ml final volume
  • Wrap in aluminum foil and store at 4°C up to three months.

100mM Sodium Thiosulfate [Na2S2O3]

  • Na2S2O3-237mg
  • Millipore water-to 50ml final volume
  • Store at 4°C up to three months

Trypsin stock

  • Trypsin [PROMEGA Modified Sequence Grade]-20ug
  • 25mM (NH4)HCO3-1ml (resuspend gently at room temperature)
  • Store at -20°C in 100ul aliquots

10% N-octyl Glucoside

  • N-octyl Glucoside [SIGMA]-250mg
  • Millipore water-to 2.5ml final volume
  • Store at room temperature

0.1% ProteasMAX stock

  • ProteasMAX [Promega, Madison, WI]- 1mg
  • 25mM (NH4)HCO3-1ml
  • Store at -20°C in 50ul aliquots (single use only)
  • Dilute to 0.01% working concetration in a final digestion reaction