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 ^oC.

4. Scrape the cells from the dish and centrifuge( 16,000xg, 4 ^oC) 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 ^oC.

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

5. Wash with lysis buffer,by centrifuging 5 minutes (16,000xg,4 ^oC), 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 ^oC 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. Add 5-10 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.