- Proteins and Peptides
- Lysates and Cell Lines
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.