TAG-LITE RECEPTOR LIGAND BINDING ASSAY

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1 TAG - L I T E TAG-LITE RECEPTOR LIGAND BINDING ASSAY PROTOCOL ASSAY PRINCIPLE The Tag-lite Ligand Binding Assay is a homogeneous alternative to radio ligand binding assays for HTS and compound profiling. It is suitable for both saturation binding assays (Kd) and competitive binding assays (Ki). At equilibrium, the fraction of labeled ligand bound to the receptor is proportional to the FRET signal recorded. From this resulting signal, binding affinities can be calculated. MATERIALS & EQUIPMENT MATERIALS NEEDED:TO PERFORM A TAG-LITE BINDING ASSAY Labeled cells Fluorescent Ligand Tag-lite Buffer (5X concentrate), 100 ml (Part# LABMED) Unlabeled ligand, to measure non-specific signal 384-well (small volume) white microplates HTRF -Certified Reader* Please contact your technical support team (htrfservices@cisbio.com) to obtain additional information or support * HTRF-approved readers using a monochromator for detection are not compatible with Tag-lite binding assays. SATURATION BINDING (KD DETERMINATION) A saturation binding assay measures total and non-specific binding of increasing concentrations of ligand under conditions of equilibrium. To perform the assay, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells and incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding. A negative control using unlabeled ligand is included to account for the non-specific binding of the labeled ligand to the receptor, non-receptor molecules, and the microplate. The fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells and a 100-fold molar excess of unlabeled ligand. The HTRF ratio obtained from this titration is the non-specific binding. The specific binding is calculated by subtracting the non-specific binding from the total binding at each fluorescent ligand concentration.

2 2 REAGENT PREPARATION AND ASSAY PROTOCOL FOR KD DETERMINATION Prepare all reagents (fluorescent ligand, unlabeled ligand, cells) in Tag-lite buffer Run all assay points in triplicate 1. Dispense into each well for both total and nonspecific binding [A1-L6, 72 wells total].** 2. Dispense into total binding wells [A1-L3, 36 wells total].** 3. Dispense into nonspecific binding wells [A4-L6, 36 wells total].** 4. Dispense dilutions [A1-L6]** into each appropriate well 5. Incubate at room temperature 6. Read on an HTRF-compatible reader* * HTRF-approved readers using a monochromator for detection are not compatible with Tag-lite binding assays. ** According to plate-map given as an example and annexed p.6 Total binding Cells 10 µl Tag-lite buffer 1X Fluorescent ligand (titration) Incubate at RT Read on an HTRF compatible reader Non specific binding Cells 10 µl Unlabeled ligand Fluorescent ligand (titration) Incubate at RT Read on an HTRF compatible reader COMPETITION BINDING (KI DETERMINATION) Competitive binding assay is performed to measure the dissociation constant, Ki. To perform the assay, the compound is titrated into a solution containing the fixed concentration of fluorescent ligand that allows 50% (Kd) to 80% of receptor binding and a fixed amount of cells.

3 3 REAGENT PREPARATION AND ASSAY PROTOCOL FOR KI DETERMINATION Prepare all reagents (fluorescent ligand, unlabeled ligand, cells) in Tag-lite buffer Run all assay points in triplicate Up to ten (10) compounds can be tested in one 384-well plate for a total of 36 wells per compound. 1. Dispense into each well. 2. Dispense or 5 μl compound dilutions[c1-c11]** into each appropriate well as shown. 3. Repeat for each compound tested. 4. Dispense * into each well. 5. Incubate at room temperature. 6. Read on an HTRF-compatible reader*. * HTRF-approved readers using a monochromator for detection are not compatible with Tag-lite binding assays. ** According to plate-map given as an example and annexed p 7. Cells 10 µl Compound (dose-response) Fluorescent ligand Incubate 2 h at RT Read on an HTRF compatible reader RECOMMENDATIONS FOR REAGENT PREPARATION Prepare working Tag-lite buffer (TLB 1X) from Cisbio Thaw the 100 ml vial of Tag-lite buffer 5X (TLB 5X), dilute it 5-fold in distilled water to prepare the TLB 1X that will be used to prepare all the reagents. Prepare cells Prepare a conical vial containing 5 ml of cold TLB 1X, Thaw labeled frozen cells (1 vial) in a 37 C water bath (manual shaking) until all the ice is thawed (1-2 min) and quickly transfer the cells by pipetting into the conical vial containing TLB 1X. Centrifuge 5 min at 1200 G. (The pellet may not be visible.) Gently remove supernatant by aspiration. (Do not pour out the supernatant.) Resuspend the pellet in 1 ml of 1X TLB, mix gently by pipetting up and down several times, add 1.7 ml of 1X TLB and mix gently by pipetting up and down several times. Step 1 Step 2 Step 3 Step 4 Step 5 Prepare a conical vial (A) containing 5 ml of cold TLB 1X Thaw labeled frozen cells (1vial) at 37 C (water bath, manual shaking) until all the ice is thawed (1-2 min) and transfer them quickly by pipeting into the vial prepared in Step 1. Centrifuge 5 min at 1200 G. Gently remove supernatant by aspiration do not pour out supernatant. Resuspend the pellet in 1 ml of 1X TLB. Mix gently by pipetting up and down several times. Add 1.7 ml of 1X TLB. Mix gently by pipetting up and down several times. Keep the cells at R.T. Be careful the pellet may not be visible.

4 4 Prepare fluorescent ligand for Kd determination (e.g. fluorescent ligand from Cisbio) Concentration of fluorescent ligand from Cisbio is indicated on the vial label. Centrifuge or reconstitute the vial then dilute the fluorescent ligand stock solution with TLB 1X to obtain the highest concentration for the saturation binding curve, starting with the F1 solution, prepare 1/2 serial dilutions in TLB 1X. (add 100 µl of F1 to 100 µl of TLB 1X, mix gently and repeat the 1/2 serial dilutions to prepare F2, to F11 solutions RECOMMENDED DILUTION PROCEDURE FOR FLUORESCENT LIGAND F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 Made from stock solution 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X 100 µl F µl TLB 1X Add 100 µl of TLB 1X from F2 to F11 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 Prepare unlabeled ligand for Kd determination Prepare a working solution of unlabeled ligand in TLB 1X at 100-fold the concentration of F1 solution Please refer to literature accompanying the unlabeled ligand for stock concentration provided Prepare compounds for Ki determination Dilute compounds with TLB 1X. Starting with the C1 solution, prepare 1/10 serial dilutions in TLB 1X, add 10 µl C1 to 90 µl of TLB 1X, mix gently and repeat the 1/10 serial dilutions to prepare C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 solutions. RECOMMENDED DILUTION PROCEDURE FOR COMPOUNDS C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 Made from stock compounds 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X 10 µl C µl TLB 1X Add 90 µl of TLB 1X from C2 to C11 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 Prepare fluorescent ligand for Ki determination For the competition dose-response of compounds, the optimal fluorescent ligand concentration is the one that allows 50% (Kd) to 80% of receptor binding. The concentration of Tag-lite fluorescent ligand from Cisbio is indicated on the vial label. Dilute compounds with TLB 1X.

5 5 Example of plate map used for KD determination A B C D E F G H I J K L F1 F2 F3 F4 F5 F6 F7 F8] F9 F10 F11 (10 μl total TLB 1X) Repeast Well G1 Repeast Well G1 F1 F2 5 μlunlabeled ligand F3 5 μlunlabeled ligand F4 10 μ L l a b e l e d c e l l s 5 μ L u n l a b e l e d l i g a n d F5 F6 F7 F8 F9 F10 F11 Repeat Well A4 Repeat Well B4 Repeat Well C4 Repeat Well D4 Repeat Well E4 Repeat Well F4 Repeast Well G4 Repeat Well H4 Repeat Well I4 Repeat Well J4 Repeat Well K4 Repeat Well L4 Repeat Well A4 Repeat Well B4 Repeat Well C4 Repeat Well D4 Repeat Well E4 Repeat Well F4 Repeast Well G4 Repeat Well H4 Repeat Well I4 Repeat Well J4 Repeat Well K4 Repeat Well L4

6 6 Example of plate map used for Ki determination A B C D E F G H I J K L Compound 1 5 μl compound [C1] 5 μl compound [C2] 5 μl compound [C3] 5 μl compound [C4] 5 μl compound [C5] 5 μl compound [C6] 5 μl compound [C7] 5 μl compound [C8] 5 μl compound [C9] 5 μl compound [C10] 5 μl compound [C11] Repeat Well G1 Repeat Well G1

7 7 DATA REDUCTION 1. Calculate the ratio of the acceptor and donor emission signals for each individual well. Ratio = Signal 665 nm Signal 620 nm x Plot the HTRF ratio versus the [fluorescent ligand] or [compound] concentrations. For more information about data reduction, please visit Example of data obtained using batch 02A for Cholecystokinin CCK2 receptor with CCK8S as reference ligand and readings taken on a PHERAstarFS with flash lamp. Results may vary from one HTRF compatible reader to another. NOTES: 1. Use of an inappropriate set-up may seriously impair results. Check that you are using the set-up for Tb donor and red ligand. 2. HTRF-approved readers using a monochromator for detection are not compatible with Tag-lite binding assays. 3. Differences in Kd values may be observed between batches of labeled cells. Variability between Kd values reported on this package insert and values calculated during your experiment may also occur. Copyright 2015 Cisbio Bioassays. All rights reserved. HTRF, Tag-lite, EPIgeneous and the HTRF logo are trademarks or registered trademarks of Cisbio Bioassays. CLIP-tag is a registered trademark of New England Biolabs, Inc. HaloTag is a registered trademark of Promega Corporation. SNAP-tag is a registered trademark of New England Biolabs, Inc. Lumi4 is a registered trademark of Lumiphore, Inc. FOR MORE INFORMATION Europe and other countries +33(0) U.S. and Canada China Japan +81-(0) Visit to find a list of our regional distributors