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

Carbonic Anhydrase and Small MoleculeBinding Assay Using a Corning ® Epic ®MicroplateProtocolThis protocol describes how to perform an Epic assay that consists of the binding of smallmolecules to immobilized bovine carbonic anhydrase (30 kDa) in a 384 well Epic microplate.Materials◗ Bovine Carbonic Anhydrase II (CA II, Sigma Cat. No. C-2522)◗ Acetazolamide (Sigma Cat. No. A6011-10G, MW = 222.2 Da)◗ Furosemide (Sigma Cat. No. F4381-1G, MW = 330.8 Da)◗ Sulpiride (Sigma Cat. No. S8010-25G, MW = 341. Da)◗ Dansylamide (Sigma Cat. No. 218898-1G, MW = 250.3 Da)◗ 4-Carboxybenzenesulfonamide (Sigma Cat. No. C11804-5G, MW = 201.2 Da)◗ Sodium Acetate (NaOAc) (Fisher Cat. No. BP333-500)◗ Dimethyl Sulfoxide (DMSO) (Sigma Cat. No. D8418)◗ 1X Phosphate buffered saline (PBS) (Sigma Cat. No. P-3813)◗ 18MW distilled, filtered water◗ 500 mL, Cellulose Acetate Filter System (Corning Cat. No. 430769)◗ 1000 mL, Cellulose Acetate Filter System (Corning Cat. No. 430517)◗ 384 well polypropylene microplate (Corning Cat. No. 3657) (“source microplate”)◗ 384 well Epic microplate (Corning Cat. No. 5041) (“assay microplate”)◗ Buffer Solutions:• 20 mM acetate buffer, pH 5.5• 1X PBS, pH 7.4• 1X PBS/0.1% DMSO binding buffer, pH 7.4Reagent Preparation20 mM Acetate Buffer, pH 5.51. To prepare 500 mL of acetate buffer, add 0.8203 g sodium acetate to ~ 485 mL 18 MWdistilled, filtered water. Stir until dissolved.2. Adjust the pH to 5.5 using ~ 6 N HCl (use NaOH if too low).3. Add the solution to graduated cylinder and adjust the volume up to 500 mL with 18 MWdistilled, filtered water.4. Filter the solution using a Corning 0.22 µm Cellulose Acetate Filter System.5. Discard the solution if any signs of microbial growth appear during storage. (Smallerquantities of acetate buffer should be prepared if assays are not run on regular basis.)

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).

Good Lab Practice1. Prior to adding protein targets to Epic ® microplates, avoid exposing microplates to airfor extended period of time. It is recommended to use the microplates within 6 hoursafter opening the pouch for best results.2. Required soaking time before taking measurements is important for stabilizing sensors,thus reducing signal variability.3. Always use the same source of reagents when running multiple assays.4. Watch out for bubbles.5. Do not spin microplate upside down once protein has been added.Furosemide Binding to Immobilized Carbonic AnhydraseFor additional product or technical information, please visitwww.corning.com/lifesciences or call 1.800.492.1110. Outsidethe United States, please call +1.978.442.2200.Corning IncorporatedLife SciencesTower 2, 4th Floor900 Chelmsford St.Lowell, MA 01851t 800.492.1110t 978.442.2200f 978.442.2476www.corning.com/lifesciencesWorldwideSupport OfficesA S I A / P A C I F I CAustralia/New Zealandt 0402-794-347Chinat 86 21 2215 2888f 86 21 6215 2988Indiat 91 124 4604000f 91 124 4604099Japant 81 3-3586 1996f 81 3-3586 1291Koreat 82 2-796-9500f 82 2-796-9300Singaporet 65 6733-6511f 65 6861-2913Taiwant 886 2-2716-0338f 886 2-2516-7500Corning and Epic are registered trademarks of Corning Incorporated, Corning, NY.All other trademarks in this document are the property of their respective owners.Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001E U R O P EFrancet 0800 916 882f 0800 918 636Germanyt 0800 101 1153f 0800 101 2427The Netherlandst 31 20 655 79 28f 31 20 659 76 73United Kingdomt 0800 376 8660f 0800 279 1117All Other EuropeanCountriest 31 (0) 20 659 60 51f 31 (0) 20 659 76 73L A T I N A M E R I C ABrasilt (55-11) 3089-7419f (55-11) 3167-0700Mexicot (52-81) 8158-8400f (52-81) 8313-8589© 2012 Corning Incorporated Printed in U.S.A. 2/12 POD CLS-AN-190