Kras G12D–cRAF Binding Assay Kit

Background

Kras is a member of the RAS protein family, which are a class of small GTPases involved in cell signaling pathways. The Ras signaling pathway regulates diverse cellular processes, including cell proliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the active GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key downstream effector of RAS. Since the frequently mutated Ras genes are associated with various human tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer treatment.

Assay Principle

The Kras (G12D)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the binding status between Kras and cRAF. Tag2-Kras (G12D) in this assay kit is loaded with GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy transfer (FRET) if Tag1-cRAF binds to the Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). 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). Blocking the Kras-cRAF binding will reduce the HTRF signal. -4510 or 858453-5700 Fax: 855-898-3979 1

Application

High throughput screening of compounds that inhibit the binding between activated Kras (G12D) and cRAF for drug discovery.

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 (such as Tecan M1000 or Tecan Spark, etc.)
• 0.5 M DTT
• Adjustable micro-pipettor
• Sterile Tips

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 Binding 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 Binding 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 Binding buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
2. Step 2. Prepare cRAF solution Thaw cRAF 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: cRAF protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the cRAF protein 480-fold (1 µL cRAF + 479 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of DTT containing Binding 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 Kras (G12D) solution Thaw Kras 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. -4510 or 858453-5700 Fax: 855-898-3979 3 Note: Kras protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the Kras protein 340-fold (1µL Kras G12D + 339 µL DTT containing Binding buffer). Add 4 µl of diluted protein solution to each well.
5. Step 5. Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody and fluorescence-labeled anti-Tag1 antibody 1:200 in DTT containing Binding buffer. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 1 µl of fluorescence-labeled anti-Tag1 antibody + 198 µl of DTT containing Binding buffer. Add 10 µl of this dye mixture to each well.
6. Step 6. Incubate the reaction at room temperature for 30 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 the HTRF signal (ratio of the fluorescent intensity at 665 mm/620 mm) 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