SEAP-fusion protein binding assay

Outline:

To estimate the binding of a certain protein to cells, a fusion protein with SEAP is generated. A defined number of cells is incubated with this fusion protein. After several wash steps and heat inactivation, substrate for alkaline phosphatase is added to the test tube. Following incubation, the OD₄₀₅ is determined as a measure of alkaline phosphatase activity, which represents protein binding.

Supplies & Equipment:

• Eppendorf tubes
• 15 ml tubes
• wet ice
• microfuge
• waterbath at exactly 65°C
• EIA reader for 96-well plates at 405 nm

Reagents:

• treatments:
  • 2 x SEAP fusion protein cond. media (>25 mU/100µl)
  • neg. control: SEAP cond. media (>25 mU/100µl)
  • neg. control for heat inactivation: MEM
• HBHA + 0.1% Tween 20
• HBS
• 3 N NaOH (will need ~ 2 ml)

Procedure:

1. Prepare incubation mixes in 15 ml tubes on ice:
   • e.g. 4 ml treatment A (1000 mU in 4 ml MEM = 10 x 100 mU in 400 µl MEM)
   • e.g. 4 ml treatment B (1000 mU in 4 ml MEM = 10 x 100 mU in 400 µl MEM)
   • 4 ml SEAP (1000 mU in 4 ml MEM = 10 x 100 mU in 400 µl MEM)
   • 4 ml MEM
2. Harvest cells, count, bring to:
   • 1,000,000 cells / ml (will need 300µl per treatment),
   • 100,000 cells / ml (will need 300µl per treatment),
   • 10,000 cells / ml (will need 300µl per treatment).
3. Aliquot cell suspension into 36 Eppendorf tubes (100 µl per tube, 4 treatments x 3 cell densities x 3)
4. Add 400 µl of the incubation mix per tube.
5. Incubate for 10 - 15 min. at room temperature.
6. Spin for 1 minute in microfuge (tubes can be left open).
7. Decant incubation mixes.
8. Wash 4 x with HBHA + 0.1% Tween 20 (pipet, spin, decant).
9. Wash 2 x with HBS.
10. Decant HBS, add 100 µl HBS, close tubes.
11. Heat inactivate endogenous alkaline phosphatase by floating tubes for 25 minutes in 65°C waterbath.
12. Add 100 µl NBT/BCIP (alkaline phosphatase substrate)
13. Vortex, then spin for 1 minute.
14. For baseline determination, set up 3 tubes with 100 µl H₂O and 100 µl NBT/BCIP
15. Incubate 1 hour.
16. Add 50 µl stop solution (3 N NaOH).
17. Pipet 100 µl of each reaction into 96-well plate.
18. Measure OD₄₀₅ with EIA-reader.

Return to: Home - LabLinks - GH-PRL-PL