Target Discovery and Validation Reviews and Protocols

66 Beaty et al.
2.3.3. Protein Staining (Silver Staining and Coomassie Staining)
1. Colloidal blue staining kit (cat. no. LC6025, Invitrogen).
2. Acetic acid, glacial (cat. no. A38-500, FIsher).
3. Methanol (cat. no. MX0475P-4, EMD Chemicals Inc.).
4. Sodium carbonate (cat. no. S-2127, Sigma).
5. Silver nitrate (cat. no. SX0205-5, EMD Chemicals Inc.).
6. Sodium thiosulfate (SX0820-1, EMD Chemicals Inc.).
7. Formaldehyde (cat. no. 2106-01, J. T. Baker).
2.3.4. Tryptic Digestion
1. Scalpel handle (cat. no. S7772-1EA, Sigma).
2. Scalpel blade (cat. no. S2646-100EA, Sigma).
3. 10 mM DTT (cat. no. 43815, Sigma).
4. 55 mM Iodoacetamide (cat. no. I-6125, Sigma).
5. 50 and 100 mM NH 4 HCO 3 (cat. no. 09830, Sigma).
6. Acetonitrile (cat. no. 9017-03, J. T. Baker).
7. 12.5 ng/µL Modified trypsin, sequencing grade (cat. no. V5111, Promega).
8. 5% Formic acid (cat. no. 06450, Sigma).
2.3.5. Reverse Phase Columns
1. Fused-silica capillary tubing for liquid chromatography 75 µm id/365 µm od, (cat.
no. TSP075375, Polymicro Technologies).
2. Kasil 1 TM (potassium/silicate solution) (cat. no. 25110, ASTRO PQ
Corporation).
3. Formamide (super pure grade) (cat. no. BP228-100, Fisher).
4. Reverse phase material for liquid chromatography SB-C18, 5–15 µm (Zorbax).
5. Reverse phase material for liquid chromatography MS218, 5 µm (Vydac,
Columbia, MD).
6. Methanol (MX0475P-4, EMD Chemicals Inc.).
7. High-pressure vessel for nano-LC column packing (cat. no. SP035, Proxeon
A/S).
2.3.6. LC-MS/MS
1. Emitters for liquid chromatography FS360-20-10-N-20; tip, 10 ± 1 µm (cat. no.
FS3602010N20, New Objective).
2. Mobile phase A (H 2 O with 0.4% acetic acid and 0.005% heptafluorobutyric
[v/v]).
3. Mobile phase B (90% acetonitrile, 0.4% acetic acid, 0.005% heptafluorobutyric
acid in water).
4. Acetonitrile (cat. no. 9017-03, J. T. Baker).
5. Heptafluorobutyric acid (cat. no. H-7133, Sigma).
6. Glacial acetic acid (cat. no. A38-500, Fisher).