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Tween-20 (Sigma Cat. # P-7949); BSA (Sigma Cat. # A-7030); ABTS Liquid Substrate Solution (Sigma Cat. # A3219); Dulbecco’s PBS [10x] (Gibco BRL Cat. #14200-075). RECOMMENDED SOLUTIONS All solutions should be at ambient temperature prior to use. PBS: Dilute 10xPBS to 1xPBS, pH 7.20 in sterile water. Wash Buffer: 0.05% Tween-20 in PBS Block Buffer: 1% BSA in PBundefined Diluent: 0.05% Tween-20, 0.1% BSA in PBundefined
ELISA microplates (Nunc MaxiSorp Prod. # 439454, or Corning Prod. # 3590)
Avidin-HRP conjugate (Sigma Cat. # A-7419)
PeproTech’s Recombinant Protein PeproTech’s Antigen Affinity Purified Polyclonal or Monoclonal Antibody PeproTech’s Biotinylated Antigen Affinity Purified Polyclonal Antibody
1. Plate preparation
1) Dilute capture antibody (polyclonal) with PBS to a concentration of 1μg/ml. Immediately, add 100μl to each ELISA plate well. Seal the plate and incubate overnight at room temperature. (Monoclonal Antibody – at least 2 μg/ml).
2) Aspirate the wells to remove liquid and wash plates 4 times. Each wash consists of adding 300μl wash buffer per well, followed by aspiration. After the last wash invert plate to remove residual buffer and blot on paper towel.
3) Add 300μl blocking buffer to each well. Incubate 1 hour at R.T.
4) Aspirate and wash plate 4 times (as in step 2).
2. ELISA protocol 1) Standard/Sample: Serial dilute standard from 0.01μg/ml to zero in diluent. Add 100μl of standard or sample to each well in triplicate. Incubate at room temperature for at least 2 hours. 2) Detection: Wash plate four times. Dilute detection antibody (biotinylated) in diluent to a concentration of 0.5μg/ml (500ng/ml). Immediately add 100μl per well. Incubate at room temperature for 2 hours. 3) Avidin-HRP Conjugate: Aspirate and wash plate 4 times. Dilute Avidin-HRP conjugate 1:2000 in diluent. Add 100μl per well. Incubate 30 min at room temperature. 4) ABTS Liquid Substrate: Aspirate and wash plate 4 times. Add 100μl of substrate solution to each well. Incubate at room temperature for color development. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm.
Reliable standard curves are obtained when O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5 minute intervals until desired O.D. readings are obtained. The typical range is 5-40 minutes. O.D. readings may vary.
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