Top : Molecular Biology : Carbohydrate : Glycolipid Binding Assay

Glycolipid Binding Assay

Source: Contributed by Pingsunjim, Pallers Lab
Date Added: Tue May 14 2002
Date Modified: Thu Apr 29 2004
Abstract: This protocol can be used for the detection of glycolipids binding to immunoprecipited protein.

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 g of antibody,400 l of H2O,400 g total protein to microcentrifuge tube.
    2. Vortex and incubate at 4celsius degree for 1 Hr.
    3. Add 10 l 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 l 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 l 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 l 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.
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