Phospho-ACC (Ser79) cellular kit
Phospho-ACC (Serine 79) kit for fatty acid metabolism research
Total AMPK cellular kit HTRF®
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
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Highly specific
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High sensitivity
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 proteins expression under a no-wash assay format.
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 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.
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.
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.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
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
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
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