Fluorescent Nucleoside Triphosphates for Single
时间:2024-04-02 12:58 来源:网络整理 作者:墨客科技 点击:次
2.1 Labeling 1.2′,3′-O-(2-Aminoethyl-carbamoyl)-adenosine-5′-triphosphate (edaATP) (Jena Biosciences) (see Note ). 2. 3′-Amino-3′-deoxyATP triethylammonium salt (aminoATP) (). 3. Cy3 N-hydroxysuccinimide ester (NHS-ester) (GE Healthcare). 4. 7-Diethylaminocoumarin-3-carboxylic acid (Invitrogen). 5. 20 mM Sodium bicarbonate, pH 8.4. 6. Dimethylformamide (DMF). 7. Tributylamine. 8. Isobutyl chloroformate. 9. HPLC system, preferably with both absorbance and fluorescence detectors. 10. Strong anion exchange (SAX) Partisphere column (0.4 × 10 cm) (Whatman). 11. 0.4 M (NH4)2HPO4 adjusted to pH 4.0 with concentrated HCl. 12. HPLC-grade Methanol. 13. HPLC-grade Acetonitrile. 2.2 Purification 1.DEAE cellulose column (2 × 30 cm). 2. Triethylamine (technical grade). 3. Glass distillation apparatus suitable for up to 500 ml and having ground glass joint. 4. Boiling chips. 5. Dry ice. 6. 2-L Buchner flask, with bung and plastic tubing on the side arm, connected to a glass-scinter gas bubbler. 7. Chromatography system (fraction collector, gradient maker, pump, etc.) at 4°C with absorbance and fluorescence detector, if possible. 2.3 Concentration 1.Rotary evaporator, equipped with a cold finger condenser and high-vacuum oil pump. 2. Methanol (highest grade available). 3. Isopropanol (technical grade). 4. Dry ice. 2.4 Characterization 1.Spectrophotometer. 2. HPLC system, preferably with both absorbance and fluorescence detectors. 3. Strong anion exchange (SAX) Partisphere column (0.4 × 10 cm) (Whatman). 4. 0.4 M (NH4)2HPO4, pH 4 with concentrated HCl. 5. HPLC-grade methanol. 6. HPLC-grade acetonitrile. 7. Fluorescence spectrophotometer. 2.5 ATPase Assay 1.MDCC-PBP (). Phosphate binding protein (A197C) from E. coli, labeled with (N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide) (Invitrogen) (see Note ). 2. Rhodamine-PBP (). Phosphate binding protein (A17C, A197C) from E. coli, labeled with 6-iodoacetamidotetramethylrhodamine (see Note ). 3. Fluorescence spectrophotometer. 4. Inorganic phosphate standard solution. 3 Methods 3.1 Synthesis of Cy3-edaATP This method is based on that described by Oiwa et al. () and gives mixed (2′,3′) isomers (Fig. b). 3.1.1 Labeling1.Mix 4 μmol Cy3 NHS-ester with 20 μmol edaATP in 20 mM sodium bicarbonate, pH 8.4, for 1 h at room temperature (see Note ). 2. Analyze the reaction mixture using HPLC to confirm the formation of Cy3-edaATP. Equilibrate a Partisphere SAX column with 0.4 M (NH4)2HPO4 with 20% (v/v) methanol: flow rate of 1 ml/min at room temperature (see Notes 4 and 5). 3. Add an aliquot of the reaction mixture (1–10 nmol) to 100 μL of the running buffer. 4. Inject the solution onto the column. 5. (责任编辑：admin) |