TR-FRET VHL Binding Assay Kit (384)

Background

Von Hippel–Lindau (VHL) is a member of an E3 ubiquitin ligase, a five-component complex including VHL, Cullin 2 (CUL2), Elongin B, Elongin C and RBX1 (RING-box protein 1). It is one of the most widely used E3 ligase recruiters in the design of PROTACs (Proteolysis-Targeting Chimeras) for targeted protein degradation (TPD) drug discovery. VHL plays a critical role in bringing the target protein and the ubiquitination machinery together for protein degradation via the proteasome.

Assay Principle

The TR-FRET VHL Binding Assy kit is designed to measure the binding affinity of VHL and its ligand, and it includes Tag1-VHL-5C (VHL/CUL2/EloC/EloB/RBX1 complex), Terbium-labeled Anti-Tag1 antibody and fluorescent labeled VHL ligand VH032. The binding of VHL to the ligand brings Terbium (fluorescence donor) on the anti-Tag1 antibody in close proximity to the fluorophore (FL) on VH032 (fluorescent receptor), which results in fluorescence resonance energy transfer (FRET). Thus, the binding status of VHL and VH032 can be quantitively determined using HTRF signal by calculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). If an compound binds to the VHL and blocks VH032 binding, the HTRF signal will be reduced.

Application

High throughput screening of compounds that bind to VHL for drug discovery.

Instrument Required

A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required. -4510 or 858453-5700 Fax: 855-898-3979 1

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• Adjustable micro-pipettor
• Sterile Tips
• Prepare the inhibitor compound solution
• Prepare VHL-5C solution
• Add inhibitor
• Prepare FL-VH032 solution
• Prepare dye solution
• Incubate the reaction at room temperature for 60 minutes.
• Measure fluorescent intensity
• Excitation wavelength at 340 nm and emission at 620 nm.
• Excitation wavelength at 340 nm and emission at 665 nm.
• Calculate sample HTRF signal of each well.
• Calculate percentage activity

Assay Protocol

1. Step 1. Prepare VHL-5C solution Thaw VHL-5C protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: VHL-5C protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the VHL-5C protein 40-fold (1µL VHL-5C + 39 µL assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.
2. Step 2. Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of assay buffer to each of negative and positive control wells. If the compound is diluted in 10% DMSO, add 2 µl of assay buffer containing 10% DMSO to each of negative and positive control wells.
3. Step 3. Prepare FL-VH032 solution Dilute FL-VH032 10-fold (1 µL FL-VH032 + 9 µL of assay buffer). Add 4 µl of diluted protein solution to each well.
4. Step 4. Prepare dye solution Dilute Terbium-labeled anti-Tag1 antibody 1:100 in assay buffer. For example: 1 µl of Terbium- labeled anti-Tag1 antibody + 99 µl of assay buffer. Add 10 µl of this dye mixture to each well. -4510 or 858453-5700 Fax: 855-898-3979 3
5. Step 5. Incubate the reaction at room temperature for 60 minutes.
6. Step 6. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
7. Step 7. Excitation wavelength at 340 nm and emission at 620 nm.
8. Step 8. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate sample HTRF signal of each well. Calculate percentage activity In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.

Assay Validation

Biological Pathway / Process

Ubiquitin-Proteasome / Targeted Protein Degradation

Therapeutic / Disease Area

Oncology; PROTAC Drug Discovery