PKMYT1 Binding Assay Kit

Background

PKMYT1, a membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, belongs to WEE kinase family that plays an important role in the regulation of mitosis. PKMYT1 is involved in cell cycle progression and in response to DNA damages by inhibition of CDK1 activity through specific phosphorylation of Tyr15 and Thr14. Overexpression of PKMYT1 is observed in both solid and hematological tumors. Therefore, PKMYT1 is considered a potential therapeutic target for cancer treatment.

Assay Principle

The PKMYT1 binding assay kit is a TR-FRET based assay, which is designed to screen compounds that bind to PKMYT1. A fluorescence-labelled tracer, that can bind to PKMYT1, and the N-terminal GST-tagged full-length human PKMYT1 are used in this assay kit. A Terbium-labeled anti-GST antibody binding to the GST-PKMYT1 serves as a fluorescence donor (HTRF donor). if the fluorescence-labeled tracer binds to the PKMYT1, the binding brings Terbium on the anti-GST antibody close to the fluorophore on the tracer (HTRF acceptor). Activation of the Terbium results in fluorescence resonance energy transfer (FRET). Thus, the binding status can be quantitively measured by calculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). The competitive binding of a non-fluorescence-labeled compound will reduce the FRET signal.

Application

High throughput screening of compounds that inhibit the PKMYT1 activity. Aurora Biolabs, LLC; www.aurorabiolabs.com; San Diego, CA, USA. Tel: 858-215-4510 or 858-453-5700 Fax: 855-898-3979 1

Instrument Required

A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• 0.5 M DTT
• Adjustable micro-pipettor
• Sterile Tips
• Prepare assay buffer containing 1 mM DTT
• Prepare the inhibitor compound solution
• Prepare PKMYT1 solution
• Add inhibitor
• Prepare fluorescence-labeled tracer and Tb-labeled anti-Tag2 antibody 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 the ratio of the fluorescent intensity of each well.
• Calculate percentage activity

Assay Protocol

1. Step 1. Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
2. Step 2. Prepare PKMYT1 solution Thaw PKMYT1 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 protein at -80°C. Note: PKMYT1 protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PKMYT1 protein 20-fold (1 µL PKMYT1 + 19 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to each positive control well and inhibitor test well. Add 8 µl of 1X DTT containing buffer to each of negative control well.
3. Step 3. Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).
4. Step 4. Prepare fluorescence-labeled tracer and Tb-labeled anti-Tag2 antibody solution Thaw the tracer and the antibody to room temperature. Dilute the tracer 100-fold and the antibody 200-fold with 1X assay buffer containing DTT. For example, add 2 µl of the tracer and 1 µl of the anti-tag2 antibody to 200 µl of 1X DTT containing assay buffer.
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 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.

Biological Pathway / Process

Cell Cycle Regulation (Mitosis entry; CDK1 inhibition)

Therapeutic / Disease Area

Oncology (solid and hematological tumors)