S.R. — 1/2004
Procedure
- 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.
- Wash 3X with TBST (pour onto plate, empty into sink, hit onto towel 3x to clear wells).
- Block plate by adding 270 µl/well of BSA/TBS solution.
- Incubate @37°C for 1 hr.
- Wash 3x withTBST.
- 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).
- Incubate @ RT for 1 hr while shaking.
- Meanwhile let TMB solution equilibrate to RT.
- Wash plate 3x and add 100 µl/well rabbit polyclonal anti-p27 antibody diluted 1:1000 in assay buffer.
- Incubate 1 hr @ RT with shaking.
- Wash plate 3x and add 100 µl/well anti-rabbit HRP conjugated antibody diluted 1:1000 in assay buffer.
- Incubate 1 hr @ RT w/shaking.
- Wash 3x and add 100 µl/well TMB. Allow to develop w/o shaking for up to 15 min. @ RT.
- Inactivate TMB with 1 N HCL and read @ A450 with TMB-S program in a plate reader.
Materials
| Carbonate Coating Buffer |
TBS |
Assay buffer |
| 25 mM sodium bicarbonate |
10 mM Tris, pH 7.4 |
PBS |
| 25 mM sodium carbonate |
8 g/L NaCl (137 mM) |
0.1% w/v BSA |
| pH to 9.7 |
0.2 g/L KCL (2.7 mM) |
0.1% v/v Tween-20
(Sigma P7949) |
TBST
TBS + 0.05% Tween 20
BSA/TBS
TBS + 2% w/v BSA
Antibodies
anti-p27 monoclonal antibody (BD Transduction Labs, 610241)
Rabbit anti-mouse p27 antibody (Fero lab)
TBM Eliza Color Substrate
3,3'5,5'-Tetramethyl-benzidine (TMB), 100 mL Sigma (T-0440)
Store at 4ºC
Plates
Nunc immuno-plate, C96 Maxisorp
Tags: Fero-Lab-Protocols, Fero-Protein
