The total AMPK kit is designed to monitor the level of cellular AMP-activated protein kinase and can be used as a normalization assay for the phospho AMPK kit.
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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
  • High sensitivity High sensitivity
The total AMPK kit is designed to monitor the level of cellular AMP-activated protein kinase and can be used as a normalization assay for the phospho AMPK kit.
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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 total AMPK enables the detection of the AMPK expression level, and is used as a normalization assay with our phospho-AMPK (Thr172) kit.

Benefits

  • VALIDATED ON ENDOGENOUS EXPRESSION
  • SPECIFICITY
  • DATA NORMALIZATION

Total-AMPK assay principle

The Total-AMPK assay quantifies the expression level of AMPK in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-AMPK assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of AMPK in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total AMPK assay princple

Total-AMPK 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 Total AMPK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total AMPK 2-plate assay protocol

Total-AMPK 1-plate assay protocol

Detection of total AMPK 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.
Total AMPK 1-plate assay protocol

Validation of Total AMPK kit in mouse and human cells

Cells were seeded in a T175 flask in complete culture medium and incubated for 2 days until reaching 80% confluency. After treatment with H2O2, cells were lysed and 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 was 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.
Human HepG2 cells treated with 15 mM H2O2.
Mouse myoblast C2C12 cells treated with 10 mM H2O2.

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

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

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 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 total Kit / 63ADK060PEG-63ADK060PEH

63ADK060PEG-63ADK060PEH - 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

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

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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