Overview

This protocol can be used for detection of gangliosides.Spicific antibodies to gangliosides are added to cells and allowed to bind to the cell surface. The nonbound antibody is washed away and an enzyme conjugated second antibody is then detected by the addition of an enzyme-spicific substrate.

Procedure

1. Trypsinize confluent flasks
2. pool and count cells
3. Resuspend to 4x100,000 cells/ml medium.
4. Add100ul/cell to 96 well culture plates
5. Incubate 24Hrs at 37c
6. Wash plates twice with PBS
7. add 150ul/well 10% Buffered Formalin
8. Fix for 20 minutes at room temperature
9. Wash three times with PBS
10. Add 250ul/well PBS:2%BSA
11. Incubate 30minutes at room temperature.
12. Wash plates twice with PBS.
13. Add 100ul/well primary antibody diluted in PBS
14. incubate for 2hr at 37c
15. Wash 5 times with PBS.
16. Add 50ul/well secondary antibody:HRP diluted in PBS.
17. Incubate for 1hr at 37c.
18. Wash 5 times with PBS.
19. TMB stoped 450nm.