Total AMPK cellular kit
Simple and robust detection kit for Phoshpo-AMPK & Total AMPK
Total ACC Cellular Kit HTRF®
This HTRF kit is designed to monitor the expression level of cellular ACC1/2, as a readout of the SREBP1c pathway activation. It can be used as a normalization assay for the phospho-ACC kit.
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All inclusive kit
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High sensitivity
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No-wash
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Low sample consumption
Overview
ACC (Acetyl-CoA carboxylase) is an important regulator of fatty acid metabolism. By catalyzing the carboxylation of acetyl-CoA to malonyl-CoA, ACC serves as a novel biomarker or drug target in diabetes and obesity research. The enzymatic activity of ACC is inhibited by phosphorylation on Ser-79 residue.
Like various genes involved in fatty acid regulation, the transcription of ACC genes is tightly regulated by the SREBP1c transcription factor. When activated, this transcription factor binds to the SRE DNA sequence upstream from the ACC gene promoter, and activates its transcription. Thus, the quantitative detection of Total ACC represents a valuable readout of SREBP1c pathway activation.
The HTRF Total ACC cellular assay monitors the expression levels of both ACC 1 and ACC2, and can be used as a normalization assay with the HTRF phospho-ACC Ser79 kit. This kit is compatible with the buffers from the phospho-ACC kit, so the same lysate can be used for analyzing both the phosphorylated and the total protein populations.
This cell-based assay enables simple yet sensitive and efficient quantification of ACC 1/2, and offers enhanced convenience over ELISA or WB assays.
Benefits
- SPECIFICITY
- PRECISION
- DATA NORMALIZATION
Total-ACC assay principle
The Total-ACC assay quantifies the expression level of ACC in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-ACC 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 the presence of ACC 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-ACC 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 the addition of Total ACC HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-ACC 1-plate assay protocol
Detection of total ACC 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.
HTRF Total ACC is a relevant readout of the SREBP1c pathway
T0901317, a synthetic LXR nuclear receptor agonist, stimulates SREBP-1c transcription. Activated SREBP1c transcription factor binds to an SRE DNA sequence upstream from the ACC gene promoter, and activates its transcription. Thus, the quantitative detection of Total ACC represents a valuable readout of SREBP1c pathway activation.
75,000 human HepG2 cells were plated in 96-well plates using DMEM culture medium, and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated with T0901317 (10µM) for 4h or 48h. Medium was then removed, and the cells were next lysed with 50 µL of lysis buffer 4 for 30 minutes at RT under gentle shaking. 16 µL of lysate were analyzed either by HTRF Phospho ACC (Ser79), Total ACC, and 4 µL with HTRF Alpha-Tubulin to normalize the assays. The HTRF signal was recorded after an overnight incubation. Normalization Tubulin was calculated as the HTRF ratio Phospho ACC (Ser79) or HTRF ratio Total ACC divided by the HTRF ratio of alpha-tubulin X 100.
T0901317 effects were analyzed using the Alpha-tubulin normalized results obtained with or without treatment.
Total ACC, companion of the Phospho ACC kit on human HepG2 cell lines treated with dorsomorphin.
100,000 human HepG2 cells were plated in 96-well plates using complete DMEM culture medium, and incubated for 24 h at 37 °C - 5% CO2. Cells were stimulated with different concentrations of dorsomorphin (2h) in SVF free DMEM culture medium. The cells were lysed with 50 µL of lysis buffer 4 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 ACC (Ser79) or Total ACC detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Total ACC HTRF vs Western Blot on human HepG2 cell line stimulated with T0901317 (10µM)
The Human HepG2 cell line was seeded in a T175 flask in complete DMEM culture medium, and incubated for 24 h at 37°C, - 5% CO2. Cells were stimulated with T0901317 (10 µM) during 16h. Medium was removed and the cells were then lysed with 3 mL of lysis buffer 4 for 30 min at RT under gentle shaking. Soluble supernatants were collected after a 10 min centrifugation.
Serial dilutions of cell lysate were performed in the lysis buffer, and 16 µL of each dilution were transferred into a 384-well sv white microplate before the addition of 4 µL of HTRF Total ACC detection reagents. Equal amounts of lysate were used for a side by side comparison between HTRF and Western Blot.
Acetyl-Coenzyme A Carboxylase ACC in fatty acid metabolism
Acetyl-Coenzyme A Carboxylase, with its two mammalian isoforms ACC-1 and ACC-2 (or ACC-alpha & ACC-beta), are ubiquitous, pivotal enzymes that are crucial for cellular energy metabolism. The ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA is attributed to ACC-1, whereas ACC-2 carboxylation of malonyl-CoA is exclusively related to mitochondrial beta-fatty acid oxidation. ACC-1 and 2 are key regulators of fatty-acid biosynthesis and oxidation, and thus present attractive targets in the design of drugs against Type 2 Diabetes, metabolic syndrome, heart disease, and even cancer.
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
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
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
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
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
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Product Insert ACC total Kit / 64NACPEG-64NACPEH
64NACPEG-64NACPEH - Product Insert
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
Safety Data Sheet (DEU) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (ELL) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (ITA) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (SPA) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) ACC total Kit / 64NACPEG
64NACPEG - Safety Data Sheet
Safety Data Sheet (DEU) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (ELL) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (ITA) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (SPA) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) ACC total Kit / 64NACPEH
64NACPEH - Safety Data Sheet
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