p27 ELISA

S.R. — 1/2004

Procedure
  1. 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.
  2. Wash 3X with TBST (pour onto plate, empty into sink, hit onto towel 3x to clear wells).
  3. Block plate by adding 270 µl/well of BSA/TBS solution.
  4. Incubate @37°C for 1 hr.
  5. Wash 3x withTBST.
  6. 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).
  7. Incubate @ RT for 1 hr while shaking.
  8. Meanwhile let TMB solution equilibrate to RT.
  9. Wash plate 3x and add 100 µl/well rabbit polyclonal anti-p27 antibody diluted 1:1000 in assay buffer.
  10. Incubate 1 hr @ RT with shaking.
  11. Wash plate 3x and add 100 µl/well anti-rabbit HRP conjugated antibody diluted 1:1000 in assay buffer.
  12. Incubate 1 hr @ RT w/shaking.
  13. Wash 3x and add 100 µl/well TMB. Allow to develop w/o shaking for up to 15 min. @ RT.
  14. 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
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109
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