The Transcreener AMPK Assay determines AMPK enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener AMPK Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The AMPK assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also make it extremely amiable to HTS.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The adenosine kinase assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized AMPK reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Both reactions have 25 μM ATP and 20 μM SAMS. Z’ shown here = 0.85.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use both 25 µM ATP with 20 µM SAMS peptide substrates. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The AMPK enzyme reaction took place for 60 minutes at room temperature. The enzymatic reaction was then halted using Stop & Detect Buffer B included in the kit and assay plates were read.
Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 0.5 µg/mL AMPK, 25 µM ATP, 20 µM SAMS for an enzyme reaction of 60 minutes at room temperature.
Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.
Assay conditions for the AMPK titration shown include 25 µM ATP, 20 µM SAMS, and 24 µg/mL ADP² antibody.
Assay conditions for the AMPK titration shown include 25 µM ATP, 20 µM AMPK, 55 nM tracer, and 8 nM ADP² Tb Antibody.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for FP is 15.5 μg/mL for 25 μM ATP. Yours may vary depending on, enzyme, and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Stops enzyme reactions requiring Mg²+. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 25 μM for AMPK. |
| Component | Notes |
|---|---|
| AMPK Enzyme | We have successfully used recombinant full-length human AMPK from Carna Biosciences Cat. #02-113 We've used AMPK at 0.5 μg/mL for screening purposes. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 5 mM MgCl2,, 0.01% Brij-35. Enzyme buffer components may be optimized as required. |
| Substrate | Use the substrate to be phosphorylated for your experiment. Here we used SAMS Peptide at 20 µM (Anaspec Cat. # AS-60167-5) |
| 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 ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-micoliter 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. |
AMPK is targeted for many diseases, including metabolism, inflammatory diseases, tumor suppression, and coronavirus. SARS-CoV-2 uses the ACE2 receptor to infect the human body. Research shows AMPK increases ACE2’s expression and stability. Once coronavirus enters the body, it downregulates ACE2, causing a chain of events leading to deadly cardio-pulmonary issues.
One drug currently on the market (Metformin) activates AMPK via phosphorylation. Currently used as a diabetes drug, Metformin has positive effects on both lipid and glucose metabolism via AMPK.
Another AMPK activator AICAR has shown to improve alveolar and capillary barriers preventing damage from an infection like SARS-CoV-2 in mice. Further work needs to be done but AMPK is a target of interest for coronavirus treatment.
The Transcreener ADP2 AMPK Assay is an excellent tool for researchers examining the therapeutic effects of AMPK.
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