p27 ELISA

Procedure

2. Dilute p27 monoclonal antibody 1:1000 (v/v) in Carbonate Coating Buffer. Add 100 µl/well and incubate o/n @ 4°C or 1 hr @37°C.
4. Wash 3X with TBST (pour onto plate, empty into sink, hit onto towel 3x to clear wells).
6. Block plate by adding 270 µl/well of BSA/TBS solution.
8. Incubate @37°C for 1 hr.
10. Wash 3x withTBST.
12. Dilute samples and standards in assay buffer and add 100 µl/well (the highest concentration of standards is added to 200 µl then serially diluted in 100 µl assay buffer on the plate).
14. Incubate @ RT for 1 hr while shaking.
16. Meanwhile let TMB solution equilibrate to RT.
18. Wash plate 3x and add 100 µl/well rabbit polyclonal anti-p27 antibody diluted 1:1000 in assay buffer.
20. Incubate 1 hr @ RT with shaking.
22. Wash plate 3x and add 100 µl/well anti-rabbit HRP conjugated antibody diluted 1:1000 in assay buffer.
24. Incubate 1 hr @ RT w/shaking.
26. Wash 3x and add 100 µl/well TMB. Allow to develop w/o shaking for up to 15 min. @ RT.
28. Inactivate TMB with 1 N HCL and read @ A₄₅₀ with TMB-S program in a plate reader.

Materials

TBST
TBS + 0.05% Tween 20

BSA/TBS
TBS + 2% w/v BSA