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        p27 ELISA

        互联网

        2413

        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

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