Phospho-ACC (Ser79) Cellular Assay Kit

Phospho-ACC detection as a marker for fatty acid metabolism in diabetes and obesity research

Cisbio's cell-based homogeneous HTRF® phospho-ACC assay (Ser79) enables an AMPK pathway readout by the quantitative detection of ACC1/2 phosphorylation on serine 79. Acetyl-CoA carboxylase constitutes an important regulator of fatty acid metabolism by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA, thus serving as a biomarker or drug target in diabetes and obesity research. Phosphorylation by AMPK at Ser79 inhibits the enzymatic activity of ACC. The phospho-ACC assay is suitable for cell-based screening of AMPK activators or modulators. The simple add-and-read protocol, eliminates all wash steps for a faster analysis and easy miniaturization while maintaining a high-quality specific output. The phospho-ACC ready-to-use kit contains all reagents you need and offers enhanced convenience over ELISA or WB.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

The phospho ACC assay is based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-ACC antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind ACC, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-ACC can be quantitatively detected using the HTRF phospho-ACC (Ser79) cellular assay kit reagents and most TR-FRET multimode plate readers.

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables the cells' viability and confluence to be monitored in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

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. ACC-1 catalyzes the ATP-dependant carboxylation of acetyl-CoA to malonyl-CoA, in fatty acid synthesis, whereas malonyl-CoA carboxylized by ACC-2 is exclusively involved in the regulation of mitochondrial fatty acid β-oxidation. ACC-1 and ACC-2 are therefore key regulators of fatty-acid biosynthesis and oxidation. ACC-1 is highly expressed in lipogenic tissues such as liver and adipose, while ACC-2 is the predominant form in oxidative tissues such as heart and skeletal muscles. When the energy level of the cell is low, due to cellular stress, exercise or fasting, AMPK (Adenosine Monophosphate-activated Protein Kinase) inactivates both ACC-1 and ACC-2 by phosphorylation at Ser79 and Ser221 respectively, switching on fatty acid oxidation and switching off fatty acid synthesis. Inversely, after food intake, secreted insulin activates PP2 (Protein Phosphatase 2) which in turn activates ACC-1 and ACC-2 by dephosphorylation.

Due to the dysregulation of ACC activity in different human disorders, this enzyme has recently become an attractive target in the design of new drugs against Type 2 diabetes, obesity, metabolic syndrome, heart diseases, but also cancer and microbial infections.

Product Performance

1. Dorsomorphin inhibition on phospho-ACC level in HepG2 liver cells using phospho-ACC (Ser79) cellular assay

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.

2. Cellular stress effect (H2O2 treatment) on phosphorylated ACC level, measured with phospho-ACC (S79) cellular assay

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.

3. Inhibition effect of dorsomorphin on the phosphorylation of ACC at serine 79, measured with phospho-ACC cellular assay

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
ACC phospho-S79 kit - 500 tests64ACCPEG
ACC phospho-S79 kit - 10,000 tests64ACCPEH
ACC phospho-S79 kit - 1 x 96 tests64ACCPET
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ACC phospho-S79 kit - 50,000 tests64ACCPEY
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Companion products

DescriptionCat. noProduct insertMSDS
ACC phospho-S79 kit control lysate64ACCTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF

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