Sandwich ELISA Protocol for Bcl3 Antibody (NB100-352)

Sandwich ELISA

1. Plate 0.1 ml of Bcl-3-6-his protein (50 ug/ml), overnight at 4C.

2. Block the plate with 0.2 ml blocking buffer, consisting of Tris buffer + NaN3 + 30% fetal calf serum, overnight at 4C.

3. Wash the wells three times.

4. Add 0.1 ml of anti-Bcl-3 supernatant [NB 100-352]and titrate down the plate in two fold dilutions.

5. Incubate for 1 hour at room temperature.

6. Wash the wells 3 times.

7. Add biotinylated- mouse anti-hamster IgG and incubate for 1 hour at room temperature.

8. Wash the wells 3 times.

9. Add extravidin-alkaline phosphatase and incubate for 1 hour at room temperature.

10. Wash the wells 5 times.

11. Add substrate (para-nitrophenyl phosphate), diluted in glycine buffer (pH 10.4).

12. Read the plate at OD405.