Dialysis (If necessary to clean up sample)
- Make dialysis buffer, 0.2M NH4HCO3, pH ~ 8.00.
- Use 8000 M.W.C.O., cut a 3.5-inch piece of dialysis membrane and cut
open along seam (for Mini-micro dialyzer) or use premade dialysis frames
for Micro-dialyzer.
- Wash dialysis tubing thoroughly with ddH2O.
- Fill the dialysis unit with dialysis buffer and secure the dialysis
membrane in place.
- Place the dialysis unit on stir plate, slowly stir.
- Dispense protein solutions into wells and dialyze for a minimum of
3 hours.
- After dialysis, dispense equal amounts to the protein solutions into
clean 1.5mL microcentrifuge tubes.
- These solutions can be frozen at -20ºC, indefinitely for use as
digestion controls.
Digestion
- Make Trypsin Solution to a concentration of [0.1ug/ul], 1 hour before
use and place on ice.
- If proteins have disulfide bonds, a reduction/alkylation process will
be necessary prior to trypsin addition.
- Add 40ul DTT solution and incubate at 56ºC for 45 mins.
- Remove samples from heat and allow to cool to room temperature
- Add 40ul of 55mM iodoacetamide solution and incubate at room temperature
for 30 mins, in the dark.
- Add trypsin to each tube at a 1:50, (protease:protein) ratio, close
the tube and gently flick to mix.
- Place the samples in 37ºC water bath for 2 hours.
- After 2 hours add an additional amount of trypsin to each sample, equal
to the first addition.
- Continue to incubate at 37ºC for 17-18 hours.
- Add 10ul of Formic Acid to solution. Alt: Add 10 ul of 10% TFA
to solution
- Store at -20ºC.
Ammonium bicarbonate (NH4HCO3) for dialysis buffer.
Fisher Scientific Cat. #A6430-500
Hydrochloric acid
Malinckrodt, AR Cat. #2612
Concentrated 12N
Apomyoglobin
Sigma Cat. #A-8673
Diluted with 1mL of 1:1 MeOH:H2O with 0.5% AcOH
Bovine Serum Albumin
Sigma Cat. #A-7030
Mixed with ddH2O to desired concentration.
Trypsin Modified sequencing grade: Dilute to 0.1 g/l by adding 200l of
buffer to vial.
Promega Cat. #V5111 E.C. 3.4.21.4
Sigma Cat. #T6567, Proteomics Grade Trypsin
Iodoacetamide (mix just prior to use)
10mg Iodoacetamide + 100ul 1M Ambic + 900ul ddH2O
DTT solution (mix just prior to use)
5ul 2M DTT stock + 895ul ddH2O + 100 ul 1M Ambic
2M DTT stock solution
154.3mg DTT/500ul ddH2O (can be stored at –20 for 4weeks.)