5. LB/carb plate (see item 3, Subheading 2.2).
6. PEG/NaCl (see item 9, Subheading 2.2).
7. PBT buffer: PBS, 0.05% Tween 20 (v/v), 0.2% BSA (w/v).
Sterile filter.
8. Ultrapure glycerol.
9. OMNImax E. coli.
2.5 Protein
Expression
and Purification
1. 2YT (see item 4, Subheading 2.2)
2. 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG). Dissolve
in H 2 O, sterile filter.
3. Lysis buffer: PBS, 1% Triton X-100 (v/v), 1.6 mM MgCl 2 ,
0.1 μg/ml lysozyme, 15 μg/ml DNase I, 1 mM PMSF.
4. Wash buffer: PBS, 30 mM imidazole, pH 7.5.
5. Elution buffer: PBS, 300 mM imidazole, 5% glycerol, pH 7.5.
6. 100 mM EDTA in PBS.
7. Ni-NTA Agarose.
2.6 Phage Selections
1. PBS.
2. Blocking solution: PBS, 0.5% BSA (w/v). Sterile filter.
3. PT buffer: PBS, 0.05% Tween 20 (v/v). Sterile filter.
4. PBT buffer (as described in item 7, Subheading 2.4).
5. PEG/NaCl (see item 9, Subheading 2.2).
6. 2YT (see item 4, Subheading 2.2)
7. OMNImax E. coli.
8. 1 Â 10
11 p.f.u./ml M13KO7 helper phage
9. 10 mg/ml tetracycline dissolved in 50% ethanol. Filter sterile
and protect from light.
10. 100 μg/ml carbenicillin
11. 30 μg/ml kanamycin.
2.7 Phage Pool
Enzyme Linked
Immunosorbent Assay
(ELISA)
1. PBS.
2. Blocking solution (see item 2, Subheading 2.6).
3. PT buffer (see item 3, Subheading 2.6).
4. PBT (see item 4, Subheading 2.6).
5. HRP-conjugated M13 antibody.
6. 3,3
0 ,5,5
0 -tetramethylbenzidine (TMB) substrate kit: TMB
solution, peroxide solution
7. 0.6 M H 2 SO 4 .
Proteomic Peptide-Phage Display of PDZ Domains
45