View This Webinar on The Transcreener Method For Screening and Profiling GTPases

Cdc42 Assay - A Transcreener GDP Assay Application

Transcreener GDP Assay directly measures GDP produced by Cdc42 (Cell Division Cycle 42). These GDP measurements allow researchers to effectively determine the activity of the Cdc42 enzyme. The assay provides a powerful tool to screen compound libraries for Cdc42 modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. Cdc42 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

Cdc42 Enzyme Reaction Cycle

Cdc42 Assay On Off Schematic

How Does This Cdc42 Assay Work?

The Transcreener Cdc42 Assay determines Cdc42 enzyme activity by directly measuring the GDP formed by the enzyme using the Transreener 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 Cdc42 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 Cdc42 assay is available with FP, FI, and TR-FRET readouts and is in an easy-to-use mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of GDP to Measure Cdc42 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener Cdc42 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

Transcreener KRAS FI Readout

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

Transcreener KRAS Assay TR-FRET Readout

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of Cdc42
  • Screen Compound Libraries for Cdc42 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled GDP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The Cdc42 assay is compatible with 96, 384, & 1536-well formats.

Transcreener Mix and Read Assay

Detection of GDP Released By Cdc42 (EDTA Concentration Dependence)

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 1 µM GTP. Linearity is shown here under initial velocity conditions when raw data is converted to GDP formed. 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 5 μg/mL GDP antibody and 2 nM GDP Alexa 633 tracer. The Cdc42 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.

Cdc42 Enzyme Titration

Enyme Titration Cdc42 Concentration Dependence

Linear Response

Linear Response Curve Cdc42 Assay Concentration Dependence

Optimal Assay Conditions: 10 nM Cdc42 for 2 hours with 10 mM EDTA achieves >150 mP assay window and <20% conversion

Detection of GDP Released By Cdc42 (Time Dependence)

The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 1 µM GTP. Linearity is shown here under initial velocity conditions when raw data is converted to GDP formed. 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 5 μg/mL GDP antibody and 2 nM GDP Alexa 633 tracer. 10 mM EDTA substrate was used based on the concentration dependent curve. The Cdc42 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.

Cdc42 Enzyme Titration

Enzyme Titration Cdc42 Time Dependence

Linear Response

Linear Response Curve Cdc42 Assay Time Dependence

Optimal Assay Conditions: 10 nM Cdc42 for 2 hours with 10 mM EDTA achieves >150 mP assay window and <20% conversion

Cdc42 Assay Services

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 Cdc42 program (right here in Madison, Wisconsin). We can provide modulator potency profiling for Cdc42, 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. 

Assay Development Services

Contact Us to Learn More

Contact us today to see if BellBrook's Cdc42 profiling services will advance your research. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

The Role of Cdc42 As a Therapeutic Target

Cdc42 is important for regulating actin cytoskeleton, cell motility, cell polarity, and cell cycle progression. It is essential for cell transformation of Ras GTPases, which have the highest mutation rates among all cancers. Overexpression or deregulation of Cdc42 correlates to several poor prognosis cancers and tumor metastasis (Murphy et al, 2021).

Cdc42 shows great potential as a therapeutic target for cancer treatment. The Transcreener GDP Cdc42 Assay is an excellent tool for researchers examining Cdc42 for therapeutic treatments.

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