300
JOHANN PETER HOSSLE
17. Filtersare wrapped in plastic wrap (e.g. Saran) then exposed with intensifying screen to X-ray film overnight at -70°C.
Subprotocol 3
lmmuno-Screening
Materials
Buffer • Coupling buffer: 0.1 M NaHC03; 0.5 M NaCl
• HCl: 0.001 M
• Saturation buffer: 0.1 M glycine; 0.1 M NaHC03; 0.5 M NaCl
• Washing buffer (pH 4.0): 0.1 M Na-acetate; 1 M NaCl
• Washing buffer (pH 8.0): 0.1 M boric acid; 1M NaCl
• PBS: 0.1 M NaCl; 2.6 mM KCl; 6.4 mM Na2HP04; 1.4 mM KH2P04; (pH
7.0)
• 10 x TBS: 0.5 M Tris-Cl (pH 8.0); 1.5 M NaCl
• TBT: TBS + lo/o (w/v) BSA; O.lo/o (v/v) Triton X-100; store at 4°C
• Enzyme-reaction buffer: 24.3 ml 0.1 M citric acid; 25.7 ml 0.2 M Na2HP04;
50 ml H20; pH adjusted to 5.0; store at 4°C
• CNBr-activated sepharose 4B: (Pharmacia) 5 g (I g 3.5 ml gel) are preswollen on a glass sinter ftlter using cold (4°C) HCl and are then washed
with total 1 litercold HCL
• Isopropyl-ß-D-thiogalacto-pyranoside (IPTG): 16 mM in H20, sterile filtered
• 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside (X-Gal): 2o/o (w/v)
in dimethylformamide
• Thimerosal: 1 o/o
• 4-chloronaphthol: 0.3o/o (w/v) in methanol; store at -20°C
• H202: 30o/o
Host strains • Lysogenic E. coli strain: BTA282(A.gtllamp3) or BNN97
• E. coli host strain: Y1088