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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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
  • High sensitivity High sensitivity
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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Overview

5' AMP-activated protein kinase (AMPK) is considered the metabolic master switch. AMPK is activated by phosphorylation on Thr172 by CaMKK2 and LKB1, among others. Upon activation, it inhibits synthesis 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. 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.

Benefits

  • SPECIFICITY
  • VALIDATED ON ENDOGENOUS EXPRESSION

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) assay principle

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) 2-plate assay protocol

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.
Phospho-AMPK (Thr172) 1-plate assay protocol

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.
Dorsomorphin inhibition assay on phospho-AMPK in HepG2 liver cells

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.
Cellular stress effect on AMPK phosphorylation in mouse C2C12 cells.

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 transpot, make AMPK an attractive target for metabolic disease research.
AMPK signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Cisbio starter packs

96-tests kits - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert AMPK P-T172 Kit / 64MPKPEG-64MPKPEH

64MPKPEG-64MPKPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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