Phospho-ACC (Ser79) cellular kit

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.

Highly specific
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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.

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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.

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VALIDATED ON ENDOGENOUS EXPRESSION
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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.

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.

Inhibition effect of dorsomorphin on the phosphorylation of ACC at serine 79

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.

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

PI3K/AKT/mTor translational control pathway - Posters

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

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Protocoles

Guides

Key guidelines to successful cell signaling experiments

Application Notes

HTRF cell-based phospho-protein data normalization

Application Notes

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Phospho-ACC (Ser79) cellular kit

Overview

Benefits

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Protocoles

Product Insert ACC P-S79 Kit / 64ACCPET

Product Insert ACC P-S79 Kit / 64ACCPEG-64ACCPEH

Guides

Application Notes

Application Notes

Insulin High Range Assay kit

Mouse TNF alpha kit

Human Apolipoprotein E kit

MAP-Tau Kit

Total AMPK cellular kit

Phospho-VASP (Ser239) cellular kit

Phospho-VASP (Ser157) cellular kit

Human Perforin-1 kit

Phospho-AKT1/2/3 (Ser473) cellular kit

Phospho-IRS1 (Ser307 mouse/Ser312 human) cellular kit

Phospho-AMPK (Thr172) cellular kit

Phospho-mTOR (Ser2448) cellular kit

Alpha-Tubulin Housekeeping standard