Transcreener GDP Assay directly measures GDP produced by RRAS (Related Rat Sarcoma Virus Oncogene Homolog). These GDP measurements allow researchers to effectively determine the activity of the RRAS enzyme. The assay provides a powerful tool to screen compound libraries for RRAS modulators to help find new therapies for disease.
The kit comes complete with the detection reagents required to measure activity. RRAS enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.
This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.
The Transcreener RRAS Assay determines RRAS enzyme activity by directly measuring the GDP formed by the enzyme using the Transcreener GDP Assay. By detecting GDP output, the assay provides a universal method to assess the activity of any GDP-producing enzyme in real time.
Transcreener RRAS Assay technology uses a simple but highly effective method that consists of an antibody selective to GDP and a far-red fluorescent tracer. GDP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The RRAS assay is available with FP, FI, and TR-FRET. It is an easy-to-use mix-and-read format. Perform your enzyme reaction, add the detection reagents, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The RRAS assay is compatible with 96, 384, and 1536-well formats.
EDTA enhances the GDP exchange and GTP hydrolysis of some GTPases. The assay conditions below are chosen to generate the best assay window and ability to study the enzyme. The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 2 µM GTP. The enzyme buffer includes 20 mM Tris pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 10 μg/mL GDP antibody and 4 nM GDP Alexa 633 tracer. The RRAS enzyme reaction took place for 60, 120, and 180 minutes. The Detection Mix was added with the substrate to start the reaction and incubated at 30°C. Readings were taken with a CLARIOstar plate reader.
Optimal Assay Conditions: 100 nM RRAS for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion
The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 2 µM GTP. The enzyme buffer includes 20 mM Tris pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 10 μg/mL GDP antibody and 4 nM GDP Alexa 633 tracer. 10 mM EDTA was used based on the concentration dependent curve. The RRAS enzyme reaction took place for 60, 120, and 180 minutes. The Detection Mix was added with the substrate to start the reaction and incubated at 30°C. Readings were taken with a CLARIOstar plate reader.
Optimal Conditions: 100 nM RRAS for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion
The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 2 µM GTP. The enzyme buffer includes 30 mM HEPES (pH 7.5), 150 mM KCl, 20 mM NaCl, 1 mM DTT, 0.01% BSA. The Detection mix included 10 μg/mL GDP antibody and 4 nM GDP Alexa 633 tracer. The RRAS enzyme reaction took place for 2 hours. The Detection Mix was added with the substrate to start the reaction and incubated at 30°C. Readings were taken with a CLARIOstar plate reader.
Optimal Conditions: 100 nM RRAS for 2 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 RRAS program (right here in Madison, Wisconsin). We can provide modulator potency profiling for RRAS, 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. We suggest using a starting GTP concentration of 1 μM for RRAS. |
| Component | Notes |
|---|---|
| RRAS Enzyme | We have successfully used R-Ras Protein: Wild-Type from Cytoskeleton, Inc. #CSRS05. An RRAS concentration of 100 nM was used for screening and SAR. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 75 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and EDTA (20, 10, 5, 2.5, or 1 mM). Enzyme Buffer components may be optimized as required. |
| EDTA | 20, 10, 5, 2.5 or 1 mM EDTA |
| 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. |
RRAS is related to RAS GTPase family oncogenes, which have high mutation rates among several poor prognosis cancers. RRAS regulates cell adhesion, cell invasion, and cell migration and enhances integrin function. RRAS mutations have been correlated to breast cancer, colorectal cancer, and melanoma (Xiao et al, 2019).
The Transcreener GDP RRAS Assay is an excellent tool for researchers examining RRAS for therapeutic treatments.
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