Western Blot

1. Run sodium dodecyl sulphate-polyacrylamide gel electrophoresis of your sample (see Note 3) and transfer to polyvinylidene fluoride (PVDF)-membrane. For initial testing, use 200 p,g of cell lysate proteins or 300 ng of pure antigen.

2. Block membrane with 5% non-fat dry milk in TBST for 1 h on a shaker.

3. Rinse membrane with 20 mM Tris-HCl, pH 7.5, 0.5 mM NaCl, 0.5% Tween (TBST).

4. Add recombinant Fab antibody (5 p,g/mL is recommended for initial experiments) to membrane in TBST with 1% non-fat dry milk, incubate 1 h at room temperature on a shaker.

5. Wash membrane three times for 5 min with TBST.

6. Add secondary antibody to membrane, for instance anti-human F(ab)2 HRP-conjugate at 1:5000 dilution in TBST with 1% non-fat dry milk, shake for 1 h at room temperature.

7. Wash membrane three times for 5 min with TBST.

8. Develop membrane with ECL Plus or ECL Advance (Amersham) and use imager or X-ray films for detection.

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