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HTRF Human and Mouse Phospho-AMPK (Thr172) Detection Kit, 500 Assay Points

The phospho-AMPK (Thr172) kit is designed for robust cell-based quantification of AMP-activated protein Kinase modulation when phosphorylated on Threonine 172.

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Feature	Specification
Application	細胞シグナル伝達
Sample Volume	16 µL

The phospho-AMPK (Thr172) kit is designed for robust cell-based quantification of AMP-activated protein Kinase modulation when phosphorylated on Threonine 172.

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Product variants

Unit Size: 96 assay points

Part #:

64MPKPET

Unit Size: 500 assay points

Part #:

64MPKPEG

Unit Size: 10,000 assay points

Part #:

64MPKPEH

Unit Size: 50,000 assay points

Part #:

64MPKPEY

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

Overview

5' AMP-activated protein kinase (AMPK) is considered metabolic master switch. AMPK is activated by phosphorylation on Thr172 by CaMKK2 and LKB1, among ors. Upon activation, it inhibits synsis of fatty acids, cholesterol, and triglycerides, and activates fatty acid uptake and ß-oxidation as well as stimulating glucose uptake. AMPK is also involved in autophagy activation through direct phosphorylation of ULK1. Revvity's cell-based homogeneous HTRF kit phospho-AMPK (Thr172) enables quantitative detection of AMP-activated protein Kinase modulation, phosphorylated at Threonine 172.

How it works

Phospho-AMPK (Thr172) assay principle

The Phospho-AMPK (Thr172) assay measures AMPK when phosphorylated at Thr172. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AMPK (Thr172) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-AMPK (Thr172) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-AMPK (Thr172) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-AMPK (Thr172) 1-plate assay protocol

Detection of Phosphorylated AMPK (Thr172) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Assay validation

Dorsomorphin inhibition of phospho-AMPK in HepG2 liver cells

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.

Cellular stress effect on AMPK phosphorylation in mouse C2C12 cells.

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.

Simplified pathway

AMPK simplified pathway

The Adenosine Mono Phosphate-activated protein Kinase (AMPK) plays a major role in regulating cellular metabolism and metabolic hormones. It is activated when phosphorylated on Thr172. AMPK was originally defined as the upstream kinase for the critical metabolic enzymes Acetyl-CoA carboxylase (ACC1 & ACC2) and HMG-CoA reductase. AMPK stimulates catabolism while inhibiting anabolic pathways and directly affects the hypothalamus to regulate appetite. The regulation of insulin synthesis, particularly its role in glucose transport, make AMPK an attractive target for metabolic disease research.

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1
Molecular Modification	Phosphorylation
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Metabolism/Diabetes NASH/Fibrosis
Unit Size	500 assay points