Procedure

A: Preparation of the cell lysate

1.Rinse a 60 mm culture dish of confluent cells with 10 mM Tris-HCl,PH 7.4,0.15 M NaCl,1 mM MnCl2 ,and 0.2 mM PMSF.

2.Lyse the cells with 0.5ml cold buffer (10 mM Tris-HCl,PH 7.4,0.15 M NaCl,1 mM MnCl2 ,3 mM PMSF,and 0.1 M Octyl glucoside)

3.Maintain constant agitation for 20 minutes at 4 ℃ .

4.Scrape the cells from the dish and centrifuge(16,000xg,4 ℃ )for 15 minutes,the supernatant is the "total cell lysate".

B : Immunoprecipitation

1.Add 4 μg of antibody,400 μl of H2O,400 μg total protein to microcentrifuge tube.

2.Vortex and incubate at 4celsius degree for 1 Hr.

3.Add 10 μl 50% protein A : Agrose,vortex and incubate for 30 minutes at 4 ℃.

4.Centrifuge the agarose solution for 5 minutes(16,000xg,4 ℃ )and discard the supernatant.

5.Wash with lysis buffer,by centrifuging 5 minutes (16,000xg,4 ℃ ),repeat wash twice.

C: The crosslinking of ganglioside

1.Add 200-400 μl 50-100 uM ganglioside (diluted in PBS from 5mM in DMSO stocking solution)to microcentrifuge tube.

2.Add 2.5x 10,000 particles of 1 uM Fluosphere beads.

3.Mix overnight at 4 ℃ with 200 μl 5mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

4.Wash with PBS,by centrifuging,repeat wash 3 times.

5.Resuspend the bead in PBS solution.

D:The detection of protein and gangliosides binding

1.Add5-0 μl Fluoro-bead to microcentrifuge tube with immunoprecipitated protein.

2.incubate 1 Hr at room temperature.

3.Wash with PBS or lysis buffer for 3 times.

4.Monitor by Fluoro-microscope.