Phospho-AMPK (Thr172) & Total AMPK Cellular Assay Kits

Phospho- and total AMPK assays for studying the metabolic master switch

Cisbio's cell-based homogeneous HTRF® kit for phospho-AMPK (Thr172) enables the quantitative detection of AMP-activated protein Kinase modulation, phosphorylated at Threonine 172. The HTRF® total AMPK assay kit is designed for the quantitative detection of total AMPK, phosphorylated and unphosphorylated, to normalize the phosphorylation status of AMPK with respect to its steady-state level in cells. The buffers of both HTRF® phospho- and total AMPK assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample. With its central role in several metabolic pathways, AMPK is a significant target in diabetes research and an important tool for oncological and cardiovascular disease studies. The simple add-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization, while maintaining a high-quality specific output and offering enhanced convenience over ELISA or WB.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total AMPK assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-AMPK antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with AMPK, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-AMPK can be quantitatively detected using the HTRF phospho-AMPK (Thr172) and total AMPK cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

The role of AMP-activated protein kinase (AMPK)

The Adenosine Mono Phosphate-activated protein Kinase (AMPK) plays a major role in the regulation of cellular lipid, protein  and glucose metabolism, and is a metabolic effect mediator of hormones such as leptin, ghrelin, adiponectin, glucocorticoids and insulin. AMPK is present in all tissues but plays an important role in brain, liver and skeletal muscles. It is activated when phosphorylated on Thr172 by upstream AMPK kinases (AMPKKs). Among these, we find the calmodulin-dependent kinase CaMKKβ  (kinase β) which phosphorylates and activates AMPK in response to increased calcium, and the tumor suppressor kinase LKB1 (liver kinase B1) which phosphorylates AMPK in response to an increase of AMP concentration in the cell after stress. Cellular stresses activate the AMPK pathway, which is highly sensitive to small changes in the intracellular [ATP]/[AMP] ratio. AMPK was originally defined as the upstream kinase for the critical metabolic enzymes Acetyl-CoA carboxylase (ACC1 & ACC2) and HMG-CoA reductase, which serve as the rate-limiting steps for fatty-acid and sterol synthesis in a wide variety of eukaryotes. AMPK and AKT pathways have opposite effects: after a meal, the insulin-activated kinase AKT inhibits AMPK by phosphorylation of its α-subunit at Ser485. Inversely, during times of energy stress, AMPK inhibits mTORC1 through the phosphorylation of TSC2 and Raptor. AMPK stimulates catabolism (glycolysis, fatty acid oxidation and mitochondrial biogenesis) while inhibiting anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis) and has a direct effect on the hypothalamus to regulate appetite. The regulation of insulin synthesis, especially its role in glucose transport into the cell and the secretion in pancreatic islet beta-cells, make AMPK an attractive target for the treatment of obesity, type-2 diabetes and associated co-morbidities. AMPK has also been implicated in a number of species as a critical modulator of aging through its interactions with mTOR and sirtuins.

Product Performance

1. Dorsomorphin inhibition on phospho-AMPK level in HepG2 liver cells using phospho-AMPK (Thr172) cellular assay

50,000 human HepG2 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubating with increasing concentrations of Dorsomorphin for 4 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-AMPK (Thr172) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

2. Cellular stress effect (H2O2 treatment) on mouse C2C12 muscle cells, measured with phospho-AMPK (Thr172) cellular assay

50,000 or 100,000 cells were plated in 96 well plates and incubated for 24 hours at 37 °C - 5% CO2. After incubating with increasing concentrations of H2O2 for 10 min, thus inducing a cellular stress, the medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-AMPK (Thr172) detection reagents were added. The HTRF signal was recorded after an overnight incubation and clearly shows increased phosphorylation of AMPK on Thr172.

3. Validation of the HTRF total AMPK cellular assay on mouse and human cell lines

Cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until reaching 80% confluency. After treatment for 10 min with H2O2, cells were lysed. For cell lysis, 3 mL of supplemented lysis buffer #4 were added for 30min at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before adding 4 µL of the HTRF total-AMPK detection reagents. HTRF signal was recorded after an overnight incubation time.

Human hepatocellular carcinoma HepG2 cells were treated with 15 mM H2O2.

Mouse myoblast C2C12 cells were treated with 10 mM H2O2.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
AMPK total kit - 500 tests63ADK060PEG
AMPK total kit - 10,000 tests63ADK060PEH
AMPK total kit - 50,000 tests63ADK060PEY
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AMPK phospho-T172 kit - 500 tests64MPKPEG
AMPK phospho-T172 kit - 10,000 tests64MPKPEH
AMPK phospho-T172 kit - 1 x 96 tests64MPKPET
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AMPK phospho-T172 kit - 50,000 tests64MPKPEY
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Companion products

DescriptionCat. noProduct insertMSDS
AMPK total kit control lysate63ADK060TDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
AMPK phospho-T172 kit control lysate64MPKTDA
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