Sandwich ELISA Protocol
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实验试剂
Add ddH2O up to 1.0L, pH to 9.5
Phosphate Buffered Saline (PBS):
Add ddH2O up to 10 L, pH to 7.2 with HCl
10% fetal bovine serum or 1% BSA in PBS. Filter before use to remove particulates.
150 mg 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, Cat. No. A-1888)
Add to 500 ml of 0.1M citric acid in ddH2 O
Aliquot 11 ml per vial and store at -200 C.
Avoid light exposure during preparation and storage.
Combine 50 ml dimethylformamide (DMF; Pierce, Cat. No. 20672) with 50 ml ddH2 O
Add 20 g sodium dodecyl sulfate
TMB (tetramethylbenzidine) Substrate Reagent Set:
实验步骤
2) Seal plate to prevent evaporation. Incubate at 4°C overnight.
2) Seal plate and incubate at room temperature for ≥ 1 hour.
3) Wash 3 times with PBS/Tween. Firmly blot plate against clean paper towels.
2) Seal the plate and incubate at room temperature for 2-4 hours or at 4°C overnight.
2) Seal the plate and incubate at room temperature for 1 hour.
3) Wash ≥ 3 times with PBS/Tween.
5. Add Avidin-Horseradish Peroxidase (Av-HRP):
2) Seal the plate and incubate at room temperature for 30 min.
3) Wash ≥ 5 times with PBS/Tween.
6. Add Substrate (ABTS for slower color development):
2) Read the optical density (OD) for each well with a microplate reader set to 405 nm.
注意事项
1) Try Capture Antibody at 1 – 10 µg/ml (general 2µg/ml).
2) Try Detection Antibody at 0.25 – 2 µg/ml (generally 1 µg/ml).
3) Titrate against each other to obtain optimal dilutions.
1) Try overnight incubations of standards and samples at 4°C.
1) If using HRP, avoid sodium azide in wash buffers and diluents, as sodium azide inhibits HRP.
2) Verify that appropriate antibody pairs were used and the activity of samples and/or standards.
2) Recombinant protein vials should be quick-spun for maximum recovery.
1) Increase stringency of washing steps by soaking plates for ~1 minute during washes.
2) Determine optimum Capture and Detection Antibody dilutions.
