UDP-SUGAR BINDING ASSAY FOR RGP |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 5X Loading buffer: | 50 mM Tris pH 6.8, 100 mM DTT, 2% SDS, sprinckle of BromoPhenol Blue, and 20% glycerol. Filter through a 45m filter |
| PROCEDURE | |
| NOTE: check the whole work area before and after work (wipe test for 3H ans 14C) | |
| NOTE: this assay utilizes purified GST-AtRGP1 protein as prepared in GST-protein fusion expression and purification. Total protein can also be used, as prepared by Protein preparation (from plants tissue), in which case >200 mg of total protein (preferably in 20 mM Tris-HCl pH 8, 1mM MgSO4) is required | |
| 1. GST-AtRGP1 is a ~70 kDa protein | |
Calculation for molarity from weight: 1 mg of GST-AtRGP1 x mmole/70,000 Da x 106 = 14.29 nmoles. Therefore, 1 mg is equal to 14.29 pmol/mL |
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| For this kind of calculations, the following conversion factor can be used to go from a 1 mg/mL protein solution to how many moles a mL of that solution represents: 1 mg/mL = 1 mM solution of a 1 kDa protein OR, 1 nmol/mL = 1 1 mg/mL for a 1 kDa protein. The conversion factor is thus: 1nmol/mL / xkDa = x pmol/mL, for a 70 kDa protein as above = 1 nmol/mL / 70 = 14.29 pmol/mL | |
| 2. Mix: 3 mL of 50 pmol of GST-AtRGP1, 1-5 mL of "50" pmol of UDP-sugar, 2 mL of 50 mM Tris pH 8, and dH2O to 20 mL | |
| (optional: add 2 mL of 10 mM MgSO4 - mostly for native proteins. GST-purified protein does not require any ion to work) | |
| 3. Incubate at RT for 3-5 mins | |
NOTE: the reaction can be performed at higher volumes/concentrations |
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| 4. Stop reaction with an 0.5 volumes of 5X loading buffer | |
| 5. Load the reaction in a SDS-PAGE gel (10%) | |
| 6. Stain with Coomassie (ON) and destain. The gel is fixed by now | |
| 7. Wash with water at least 2X | |
| 8. Incubate with Fluoro-Hance (1M Sodium salicylate, pH 6) | |
9. Dry gel. Expose to film |
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