General Protocol - ELISA
1. Dilute protein in Bi-Carbonate solution: Bi-Carbonate solution is made by dissolving one capsule of Carbonate-Bicarbonate Coating Buffer Capsule (Sigma C-3041) in 100ml of ddH2O to make a 50 mM buffer, pH 9.6. Load 50ml of diluted protein into each well.
2. Incubate plates at 4°C overnight OR 3hr at RT OR at 37°C for 1 hr.
3. Rinse out the plates with 1 X PBS and block with 1% BSA/PBS Add 200ml to each well.
1% BSA/PBS must be made right before use OR it can be prepared and stored at -20°C (frozen) till use.
4. Incubate plates at 4°C overnight OR 3hr at RT OR at 37°C for 1 hr. Freeze at -70°C till use.
5. Rinse out the plates 3 times with PBS.
6. Add 50ml of hybridoma supernatant to each well, or use serial dilutions of antiserum if determining antiserum titer.
7. Incubate on rocker at room temperature for 1-3 hours at room temperature.
8. Wash the plates 3 times with PBS.
9. Add 50ml of 1:1000 diluted alkaline phosphatase conjugated anti-mouse antibody to each well.
The alkaline phosphatase-conjugated secondary antibody is diluted in 1X PBS. Incubate on shaker at room temperature for 1hr.
10. Wash the plates 3 times with PBS.
11. Add 50ml of pNPP substrate solution per well.
To make the developing buffer start with: 50 ml of Diethanolamine, 258ml 1M MgCl2, add ddH20 to ~500 ml, and adjust the pH to 9.8 then add ddH2O to final 515 ml.
p-Nitrophenol Phosphate (pNPP) substrate: Sigma N-2765. Dissolve 1mg per every 1 ml of solution.
|
|
