Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 65
2.2.10. Purification of Ditags (Step 10)
1. TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0.
2.2.11. Ligation of Ditag s to Form Concatemers (Step 11)
1. 40% Polyacrylamide: 37.5:1 acrylamide:bis (cat. no. 161-0148, Bio-Rad).
2.2.12. Ligating Concatemers in pZero (Step 12)
1. SphI restriction enzyme (cat. no. 182S, New England Biolabs).
2. PZero plasmid (cat. no. K2500-01, Invitrogen).
2.2.13. Electroporation of Ligation Products and Colony PCR (Step 13)
1. ElectroMAX DH10B competent cells.
2. Zeocin (cat. no. R250-01, Invitrogen).
3. Glass beads.
4. Agarose (Select Agar cat. no. 30391-023, Invitrogen).
2.3. Mass Spectrometric Analysis of Proteins
2.3.1. Preparation of Pancreatic Juice and Pancreatic Tissue
1. Protease inhibitor tablets (cat. no. 1697498, Roche Diagnostics).
2. DC Protein Assay kit I (cat. no. 500-0111, Bio-Rad).
3. Scalpel handle (cat. no. S7772-1EA, Sigma).
4. Scalpel blade (cat. no. S2646-100EA, Sigma).
5. Phosphate-buffered saline (PBS) tablets (cat. no. P4417-100TAB, Sigma).
6. Modified radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 7.4, 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 1 mM
EDTA).
7. Sodium chloride (cat. no. S9888, Sigma).
8. Deoxycholic acid (sodium deoxycholate) (cat. no. D6750, Sigma).
9. NP-40 (cat. no. 492015, Calbiochem).
10. Trizma base (cat. no. T6066, Sigma).
11. EDTA (cat. no. E4884, Sigma).
12. Sonicator (Sonifier 250, Branson; or similar).
2.3.2. Fractionation by 1D Gel Electrophoresis
1. XCell SureLock (cat. no. EI0001, Invitrogen).
2. 10% NuPAGE, Bis-Tris gel, 1.5 mm, 10-well (cat. no. NP0315BOX, Invitrogen).
3. 4–12% NuPAGE, Bis-Tris gel, 1.5 mm, 10-well (cat. no. NP0335BOX,
Invitrogen).
4. 10X NuPAGE sample reducing agent (cat. no. NP0009, Invitrogen).
5. 4X NuPAGE LDS sample buffer (cat. no. NP0007, Invitrogen).
6. NuPAGE antioxidant (cat. no. NP0005, Invitrogen).