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