The Transcreener GDP Assay provides a unique approach to quantify activity of KRAS G12V and stimulation of KRAS G12V by its GAP and GEF counterparts. The assay uses a highly specific GDP antibody and far-red tracer to directly detect GDP produced by KRAS G12V. The novel direct detection format simplifies the protocol, reduces the risk of false positives or missing a hit, and minimizes compound interference. Our three readout options (FP, FI, and TR-FRET) allow researchers to choose the best assay format based on their plate reader compatibility and preference:
The KRAS G12V assay kit comes complete with the detection reagents required to measure activity. KRAS G12V enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.
The enzyme reaction begins with the addition & incubation of KRAS G12V enzyme and GTP substrate. Transcreener GDP Antibody & Tracer are then added & incubated. A compatible plate reader reads the plate.
Z’ data confirms the robustness of the KRAS G12V assay. A robust assay is necessary to ensure accuracy and efficiency in order to scale up for high-throughput screening. The Z’ shown here demonstrates the assay is suitable for HTS. Z’ = 0.82.
Here, our components in the 20 µL reaction included 30 mM HEPES (pH 7.5), 0.01% BSA, 150 mM KCl, 20 mM NaCl, 1 mM DTT, and 0.2 µM GTP. Reactions were run at 30°C for 3 hours. These assay conditions are chosen to generate the best assay window and ability to characterize the enzyme. Raw polarization signal (mP) is converted to GDP formed using a standard curve.
Here, our components in the 20 µL reaction included 60 nM KRAS G12V, 30 mM HEPES (pH 7.5), 0.01% BSA, 150 mM KCl, 20 mM NaCl, 1 mM DTT, and 0.2 µM GTP. Reactions were run at 30°C for 3 hours.
Interested in moving your program forward, but don’t want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your KRAS G12V program (right here in Madison, Wisconsin). We can provide modulator potency profiling for KRAS G12V, along with a variety of other GTPase enzymes, related GAPs, GEFs, and other proteins. Get accurate IC50 results fast to understand how your lead molecule interacts with other GTPases.
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| Component | Notes |
|---|---|
| GDP Antibody | Antibody specific to the nucleotide GDP. Yours may vary depending on enzyme and buffer composition. |
| GDP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| GTP and GDP | GTP and GDP are used to create the GTP/GDP standard curve. GTP is used as a phosphate donor to start the reaction. |
| Component | Notes |
|---|---|
| KRAS G12V Enzyme | We have successfully used recombinant KRAS G12V Protein from Cytoskeleton Cat. #CS-RS04. |
| Enzyme Buffer | 30 mM HEPES (pH 7.5), 0.01% BSA, 150 mM KCl, 20 mM NaCl, 1 mM DTT |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the GDP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
