eIF4E/eIF4G Binding Assay Kit

Background

Eukaryotic translation initiation factor eIF4E, the mRNA cap-binding protein, is considered the rate- limiting factor in translation. It plays an important role in cap-dependent translation initiation and recruitment of mRNA to ribosomes. Overexpression of eIF4E has been documented in numerous human cancers and contributes to transformation, tumorigenesis, and progression of cancers. Therefore, eIF4E is an attractive drug target for cancer treatment.

Assay Principle

The eIF4E binding assay kit, a TR-FRET based assay, is designed to screen compounds that bind to eIF4E. A fluorescence-labelled tracer and the N-terminal tagged full-length human eIF4E/eIF4G complex are used in this assay kit. A Terbium-labeled antibody binding to the tag on eIF4E serves as a fluorescence donor (HTRF donor). The binding of the fluorescence-labeled tracer to the eIF4E brings Terbium on the anti-Tag 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 compound will reduce the FRET signal.

Application

High throughput screening of compounds that bind to eIF4E/eIF4G.

Instrument Required

Aurora Biolabs LLC, San Diego, CA 92121, USA; www.aurorabiolabs.com; 1 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 eIF4E/eIF4G solution
• Add inhibitor
• Prepare fluorescence-labeled tracer
• 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 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 eIF4E/eIF4G solution Thaw eIF4E/eIF4G 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: eIF4E/eIF4G protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the eIF4E/eIF4G protein 200-fold (1 µL eIF4E/eIF4G + 199 µL 1X assay buffer containing DTT). Add 8 µl of diluted protein solution to each positive control wells and inhibitor test wells. Add 8 µl of 1X DTT containing buffer to each of negative control wells.
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 negative and positive control wells. Incubate at room temperature for 30 minutes (optional).
4. Step 4. Prepare fluorescence-labeled tracer Thaw the tracer at room temperature. Dilute the tracer 125-fold (1 µL of 1 M tracer + 124 µL 1X assay buffer containing DTT). Add 5 µl of diluted tracer to each well.
5. Step 5. Prepare dye solution Dilute Terbium-labeled anti-Tag antibody 1:200 (1 µl of Terbium-labeled anti-Tag antibody + 199 µl of 1X DTT-containing assay buffer). Add 5 µl of this dye mixture to each well. Dilute just enough of the antibody for each reaction set. Store remaining undiluted antibody at -80°C. Do not re-use the diluted antibody.
6. Step 6. Incubate the reaction at room temperature for 60 minutes.
7. Step 7. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
8. Step 8. Excitation wavelength at 340 nm and emission at 620 nm.
9. Step 9. 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

Cap-Dependent Translation Initiation

Therapeutic / Disease Area

Oncology (eIF4E-overexpressing tumors)