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

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).