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UA-Glo® AMP Assay Kit

UA-Glo® AMP Assay Kit

Catalog Number: UA079024 Brand: UA BIOSCIENCE
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Regular price $816 USD
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Product Details

Product Specification


Synonyms AMP检测试剂盒
Stability & Storage

Dry ice transportation. Store in the dark at -20℃ or below. Valid for 12 months.

Background

The UA-Glo® AMP Assay Kit enables homogeneous quantitative measurement of the activity of any enzyme that generates AMP in enzymatic assays, such as cAMP-specific phosphodiesterase (PDE), aminoacyl-tRNA synthetase, T4 DNA ligase, and ubiquitin ligase. This kit determines enzyme activity by quantitatively detecting the amount of AMP produced in the enzymatic reaction: the amount of AMP is positively correlated with enzyme activity, and the assay can quantitatively measure AMP generated in the presence or absence of ATP during the reaction. The kit can be used for high-throughput homogeneous screening of inhibitors targeting AMP-producing enzymes, such as PDE. The AMP detection assay offers broad applicability, high detection sensitivity, a wide dynamic range, and strong resistance to compound interference.

Components

The AMP Assay Kit consists of two main components: the AMP GR Reagent and the AMP Detection Reagent, and also includes one vial each of ATP and AMP reagents.
The AMP GR Reagent is used to terminate the enzyme reaction, remove ATP, and convert AMP to ADP.
The AMP Detection Reagent is used to convert ADP to ATP, generating a luminescent signal through the reaction of luciferase with ATP.
The kit specifications are as follows:

Size

Contain

96-well reaction number

384-well reaction number

1,000 assays

AMP GR试剂 5mL

AMP检测试剂10ml

ATP (10mM, 0.5mL)

AMP (10mM, 0.1mL)

200

1,000

10,000 assays

AMP GR试剂 50mL

AMP检测试剂100ml

ATP (10mM, 5mL)

AMP (10mM, 1mL)

2,000

10,000


Protocol

1. Enzyme Reaction Setup:

  1. Perform the enzyme reaction in a white opaque 96-well or 384-well assay plate. The recommended reaction volume is 25 µL for 96-well plates and 5 µL for 384-well plates. Test compounds with concentration gradients may be added to the enzyme reaction.

  2. Enzyme and substrate concentrations should be optimized for different enzymatic reactions. To achieve an appropriate signal-to-noise ratio, use enzyme concentrations within the linear response range. Due to the high sensitivity of the AMP Assay Kit, the amount of enzyme required can be significantly reduced.

  3. If the enzyme reaction requires ATP, use high-purity ATP for optimal results. Some commercially available ATP contains residual ADP, which may lead to high background due to the high sensitivity of the AMP Assay Kit. It is recommended to use the ATP provided in the kit or other high-purity ATP, such as Sigma-Aldrich ATP (Cat# A2383, purity ≥99%) or equivalent.

  4. The enzyme reaction may be performed using buffer systems and cofactors reported in the literature or optimized for specific experimental conditions.

  5. The temperature and duration of the enzyme reaction should be set according to the specific enzyme. For high-throughput compound screening, it is recommended to optimize the reaction at room temperature (22°C–25°C) to ensure temperature uniformity during AMP detection.

  6. No additional reagent is required to terminate the enzyme reaction after incubation. If a termination reagent is necessary for specific experimental requirements, avoid using magnesium chelators such as EDTA. The AMP detection reaction requires magnesium ions, and the final concentration must be at least 5 mM.

2. Conversion of AMP to ADP and ATP Removal After Enzyme Reaction:

  1. Take out the AMP GR Reagent and equilibrate it to room temperature (22°C–25°C). Mix gently by swirling [Note 1, 2, 3].

  2. If the enzyme reaction was performed at a non-room temperature (e.g., 30°C), equilibrate the assay plate to room temperature [Note 4].

  3. Add 25 µL of AMP GR Reagent to the 25 µL reaction in a 96-well plate (total volume 50 µL), or 5 µL of AMP GR Reagent to the 5 µL reaction in a 384-well plate (total volume 10 µL). Mix thoroughly by shaking [Note 5].

  4. Incubate at room temperature for 40 minutes.

3. Enzyme Activity Measurement:

  1. Take out the AMP Detection Reagent and equilibrate it to room temperature. Mix gently by swirling [Note 4].

  2. Add 50 µL of AMP Detection Reagent to the 50 µL mixture in the 96-well plate, or 10 µL of AMP Detection Reagent to the 10 µL mixture in the 384-well plate. Mix thoroughly by shaking. Incubate in the dark at room temperature for 30 minutes.

  3. Luminescence signals can be read 30–180 minutes after adding the AMP Detection Reagent, or even longer [Note 6].


Guidelines

1)After first use, the reagent should be aliquoted and stored protected from light at -20°C or below to ensure stability.
2)For long-term storage at -20°C, the AMP Detection Reagent may form a small amount of precipitate after thawing to room temperature. The supernatant can be used directly, or the precipitate can be removed by centrifugation before use.
3) Mixing different batches is not recommended.
4)The luciferase reaction in the AMP Detection Reagent is sensitive to temperature changes. Both the reagent and test samples/assay plates must be equilibrated to room temperature (22°C–25°C), and the temperature should remain constant (±1°C) during the testing process.
5)Unless validated, changing the recommended reagent volumes is not advised. The volume ratio of the enzyme reaction, AMP GR Reagent, and AMP Detection Reagent should be maintained at 1:1:2.
6) The luminescence signal is highly stable and remains essentially unchanged for up to 3 hours.
7)This product is intended for research use only.

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