Carbonic Anhydrase and Small Molecule Binding Assay Using a ...

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1X Phosphate Buffered Saline (PBS), pH 7.41. To prepare 1000 mL of solution, add contents of one packet (Sigma Cat. No. P-3813) toa 1L graduated cylinder and adjust volume to 1000 mL with 18 MW distilled, filteredwater. Stir until dissolved.2. Filter the solution using a 1000 mL Corning ® 0.22 µm Cellulose Acetate Filter System.1X PBS/0.1X % DMSO Binding Buffer, pH 7.41. To prepare 1000 mL of Binding Buffer, add 1 mL DMSO to a 1L graduated cylinder andadjust volume to 1000 mL with 1X PBS, pH 7.4.2. Filter the solution using a 1000 mL Corning 0.22 mm Cellulose Acetate Filter System.Bovine Carbonic Anhydrase II1. Stock Solution: Make a 2.5 mg/mL stock solution of CA II with appropriate volume of18 MW distilled, filtered water to a vial of CA II. Centrifuge at 12,000 rpm for 5 minutesat 4°C. Make small aliquots and store at -20°C. For a full microplate assay as describedin the SOP, make 300 mL CA II aliquot.2. Working Solution: Prepare a 100 µg/mL solution of CA II in 20 mM acetate buffer,pH 5.5 by mixing 300 µL of 2.5 mg/mL CA II and 7.2 mL of 20 mM acetate buffer.Working solutions should be freshly prepared for each assay.Small Compounds1. Stock Solution: Prepare 20 mM stock solution of small compounds by dissolving thecompounds in 100% dimethyl sulfoxide (DMSO). For example, dissolve 33.1 mg of furosemidein 5 mL of 100% DMSO in a 15 mL conical tube (mM = (1000 * mg/MW)/mL).Make small aliquots and store at -20°C.2. Working Solution: Prepare a 20 µM working solution of a small compound in 1X PBS.For example, mix 5 µL of 20 mM furosemide and 5 mL 1X PBS in a 15 mL conical tube.Working solutions should be freshly prepared for each assay.3. Preparation for a Dilution Series: Prepare compound working solution as highestconcentration for the dilution series, then do a 1:2 serial dilution using Binding Buffercontaining 0.1% DMSO.Carbonic Anhydrase Immobilization1. Add 15 mL of 100 mg/mL CA II in 20 mM acetate buffer, pH 5.5 into the appropriatewells of a Corning Epic ® microplate. Leave a column as buffer controls by adding 15 µLof 20 mM acetate buffer, pH 5.5. These buffer controls are important for calculatingimmobilization level (see microplate map below).
2. Centrifuge the Epic ® microplate at 800 rpm for 1 minute to remove any air bubbles.3. Seal microplate and place it at 4°C overnight.4. The next morning, remove seal and discard the solutions from all wells.5. Rinse the wells by adding 25 mL 0.1% DMSO in 1X PBS, pH 7.4.6. Discard the 0.1% DMSO in 1X PBS solution from all wells.7. Repeat the previous 2 steps (Steps 5 and 6) four times using fresh 0.1% DMSO in 1XPBS, pH 7.4.8. Add 15 mL Binding Buffer (0.1% DMSO in 1X PBS, pH 7.4) to all wells.9. Allow the microplate to soak in Binding Buffer for 2 hours (0.5 hours in the instrument)prior to performing the binding assay.Small Molecule Source Microplate Preparation1. Transfer 30 mL of the serial diluted small molecule compounds to each well of a 384 wellpolypropylene microplate (see microplate map below).Performing Binding Assay1. After 1.5 hours of soaking, load the lidded assay microplate with immobilized CA II intothe Epic reader. Allow thermal equilibration inside the reader for at least 30 minutes.2. Take baseline measurements: 6-minute readings with 1.5-minute intervals (4 data points).3. Transfer 15 mL small molecule compounds from the source microplate to the assaymicroplate.4. Pipet up and down 10 times to mix.5. Load the microplate back into the Epic reader. Allow 30 minutes thermal equilibrationinside the reader.6. Take final read: 6 minutes reading with 1.5-minute intervals (4 data points).
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