• Transcreener ADP² FI Assay Technical Manual
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Features:

• EZ Protocol
• 24 Hour Signal and Deck Stability
• Excellent Z' Values at Initial Velocity Conditions
• Compatibility with Academic and HTS Instrumentation

The Transcreener® ADP² Fluorescent Intensity (FI) Assay extends the Transcreener® platform for ADP detection by utilizing a simple fluorescent intensity output which can be used on both fluorescence readers typically found in academic and therapeutic research labs as well as more complex multimode plate readers more commonly used in core facilities and HTS labs. The Transcreener® ADP² FI Assay is a red, competitive fluorescence intensity assay based on the detection of ADP and therefore is compatible with any enzyme class that produces ADP, including protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases and glutamine synthetase. It is a simple one step homogenous detection assay, and is flexible with regard to ATP concentration (0.1 to 1,000 µM ATP). The assay provides excellent signal at low substrate conversion, with a Z' ≥ 0.7 at 2.5% ATP conversion using 1 µM ATP.

The Transcreener ADP² FI Assay was developed to follow the progress of any enzyme that produces ADP. The Transcreener ADP Detection Mixture comprises quenched ADP Alexa594 Tracer bound ADP² Antibody conjugated to IRDye® QC-1 quencher. The tracer is displaced by ADP, the invariant product generated during an enzyme reaction. The displaced tracer becomes un-quenched in solution leading to an increase in fluorescence intensity.

• UNIVERSAL: Homogenous ADP detection
• FLEXIBLE: Any ADP-producing enzyme and substrate.
• RELEVANT: Rapid pharmacologically relevant data.
• SENSITIVE: Z' ≥ 0.7 at low ATP conversion.
• SMART: One set of reagents reduces time and expense.

EZ Protocol - No More Antibody Optimization

One assay and one equation for any ATP concentration from 0.1 to 1,000 µM ATP. Determine the optimum antibody concentration for the amount of ATP in your enzyme reaction using an equation of line, then simply titrate your enzyme and proceed to your screen.

Figure 1. The relationship between ATP concentration and ADP² Antibody concentration is linear with the FI Assay. For simplicity, the line is shown out to 100 µM ATP, though linearity holds true to 1,000 µM ATP. Therefore, the quantity of the ADP² Antibody-IRDye® QC-1 for enzyme reactions that use between 0.1 and 1,000 µM ATP can be determined using the equation y = 0.93x + 07, where x = [ATP] (µM) in the enzyme reaction and y = amount of ADP² Antibody-IRDye® QC-1 (µg/mL) in the ADP Detection Mixture, m (slope) = 0.93 and b (y-intercept) = 0.7).

24 Hour Signal and Deck Stability

Excellent deck and signal stability provide flexibility and assay consistency when conducting large compound library screens.

Figure 2. Excellent signal and deck stability for up to 24 hours.

Excellent Z' Values at Initial Velocity Conditions

The sensitivity of the Transcreener ADP² FI Assay results in excellent Z' Values at low ATP conversion, enabling assay detection at initial enzyme velocity conditions.

Table 1. Excellent Z' Values at Low ATP Conversion. Z' values ≥ 0.7 were achieved at 1% ATP conversion for 10 µM, and 100 µM ATP and 5% conversion for 0.1 µM and 1 µM ATP.

Compatibility with Academic and HTS Instrumentation

Initial experiments have been performed on the Tecan Safire²™ and the Molecular Devices SpectraMax M2. Each instrument is representative of HTS and academic instrumentation, respectively.

A) PKA Titration Using SpectraMax M2 and Tecan Safire²™

Figure3A. PKA was titrated (1:2). Reactions were performed in a 10 µL assay volume with 10 µM ATP and 50 µM kemptide substrate and incubated for one hour. Detection Mixture (10 µL) was added and reactions equilibrated for one hour before reading.

B) PKA Inhibitor Dose Dependency Curves

Figure 3B. Dose dependency curves were generated. Staurosporine, H-89, and GO-6983 (known PKA inhibitors) and Tyrphostin AG1478 (a no inhibition control for PKA) were serially titrated into the PKA reaction following the assay conditions noted previously. The IC₅₀ values using both instruments (Safire²/SpectraMax M2) for staurosporine, H-89 and GO-6983 were 37/32 nM, 165/124 nM and 9.0/4.7 µM, respectively.

For more information on the Transcreener ADP² FI Assay contact BellBrook Labs:
866.313.7881 in North America
info@bellbrook labs.com

IRDye® QC-1 quencher is licensed from LI-COR®.