Author:             pingsunjim
Source:             Contributed by Pingsunjim
Date Added:     Tue May 14 2002
Date Modified: Wed Apr 28 2004

--------------------------------------------------------------------------------
 Abstract: 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.

--------------------------------------------------------------------------------

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

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