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HTRF Human and Mouse Phospho-ACC (Ser79) Detection Kit, 10,000 Assay Points
The phospho-ACC 1/2 (Ser79) kit enables the quantitative cell-based detection of ACC phosphorylation on serine 79, and is a readout for AMPK pathway activation.
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 & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Product information
Overview
Acetyl-CoA carboxylase (ACC 1/2) 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. Phosphorylation by AMPK at Ser-79 inhibits ACC enzymatic activity. This cell-based assay enables simple yet sensitive and efficient quantification of ACC 1/2 phosphorylation on Serine 79, and offers enhanced convenience over ELISA or WB assays.
Specifications
| Assay Points |
10000
|
|---|---|
| Assay Target Type |
Kit
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Therapeutic Area |
Metabolism/Diabetes
NASH/Fibrosis
|
| Unit Size |
10,000 Assay Points
|
Video gallery
How it works
Phospho-ACC (Ser79) kit assay principle
The Phospho-ACC (Ser79) assay measures ACC when phosphorylated at Ser79. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-ACC (Ser79) 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 proteins phosphorylation state under a no-wash assay format.
Phospho-ACC (Ser79) kit 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-ACC (Ser79) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-ACC (Ser79) Kit 1-plate assay protocol
Detection of Phosphorylated ACC (Ser79) 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 on phospho-ACC level in HepG2 liver cells
50,000 human HepG2 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated with dorsomorphin (2h). Medium was then removed and cells were lysed with 50µl of lysis buffer for 30min 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) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Cellular stress effect on phosphorylated ACC
50,000 HEK293 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated with H2O2 (10min), to simulate a cellular stress. Medium was then removed and cells were lysed with 50µl of lysis buffer for 30min 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) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Inhibition of dorsomorphin on ACC phosphorylation in NIH3T3 cells
50,000 mouse NIH-3T3 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated with different concentrations of dorsomorphin (2h). Medium was removed and cells were lysed with 50µl of lysis buffer for 30min 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) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Simplified pathway
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 that against Type 2 Diabetes, metabolic syndrome, heart disease and even cancer.
Resources
Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
SDS, COAs, Manuals and more
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