FUCOSYL TRANSFERASE ASSAY |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. 0.5 M Pipes-KOH pH 6.2 (1 L): | Mix 151.2 g of Pipes, 800 mL of dH2O, pH to 6.2 w/ KOH, and add dH2O to 1 Liter (Autoclave) |
| 2. 10 mg/mL tamarind XG: | Commercially available |
| PROCEDURE | |
| 1. Premix the following for 10 assays: 100 mL of 0.5 M Pipes-KOH pH 6.2, 1.0 mL of GDP-3H-fucose (NEN #989, 20mM, 5.1 mCi/mmol or equivalent), and 100 mL of 10 mg/mL tamarind XG | |
| 2. Pipette 20 mL of 10X assay pre-mix to eppendorf tubes. Add 180 mL ofprotein prep (containing 0.1% Triton X-100). Up to 60 samples can be done at one time | |
| 3. Incubate for 20-30 mins at RT (pipetting 30 assays takes about 10 minutes) | |
4. Add 0.8 mL of Ethanol (final concentration = 70%), mix and place on ice for 1-2 hours or overnight |
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| 5. Centrifuge at 14,000 rpms for 15 mins at 4oC, discard supernatant by aspiration, wash pellet with 1.2 mL 80% Ethanol, spin for 3-4 mins and aspirate supernatant. Repeat wash one more time | |
| 6. Add 100 mL of dH2O and spin for 5 secs. Let sit at RT for 5 mins and resuspend with pipette | |
| 7. Transfer content to individual reactions to scintillation vials and add 2 mL of scintillation fluid per vial | |
| 8. Count on a scintillation counter | |