Figure 2. The relationship between the protein concentration of lysates from untreated and H2O2-treated Hep G2 cells and the absorbance at 450 nm using the PathScan® Total ACC Sandwich ELISA Kit #7996 is shown.
Figure 1. Treatment of Hep G2 cells with H2O2 stimulates phosphorylation of ACC at Ser79, detected by the PathScan® Phospho-ACC (Ser79) Sandwich ELISA Kit #7986, but does not affect the levels of total ACC detected by PathScan® Total ACC Sandwich ELISA Kit #7996. Hep G2 cells (80-90% confluent) were treated 10 mM hydrogen peroxide for 10 minutes and lysed with #7018. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Acetyl-CoA Carboxylase (C83B10) Rabbit mAb #3676 (left panel) or Phospho-Acetyl-CoA Carboxylase (Ser79) Antibody #3661 (right panel) are shown in the bottom figure.
|7996C||1 Kit (96 assays)|
|Product Includes||Volume||Solution Color|
|Acetyl-CoA Carboxylase Mouse mAb Coated Microwells||96 tests|
|Acetyl-CoA Carboxylase 1 Rabbit Detection Ab||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
The PathScan® Total Acetyl-CoA Carboxylase (ACC) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of ACC. An ACC mouse antibody has been coated onto the microwells. After incubation with cell lysates, ACC (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, an ACC rabbit detection antibody is added to detect captured ACC protein. Anti-rabbit IgG, HRP-linked Antibody #7074 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total ACC.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
revised January 2016
Protocol Id: 205
PathScan® Total Acetyl-CoA Carboxylase Sandwich ELISA Kit #7996 detects endogenous levels of total ACC protein (see Figure 1). The kit sensitivity is shown in Figure 2.
Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA (1). It is the key enzyme in the biosynthesis and oxidation of fatty acids (1). In rodents, the 265 kDa ACC1 (ACCα) form is primarily expressed in lipogenic tissues, while 280 kDa ACC2 (ACCβ) is the main isoform in oxidative tissues (1,2). However, in humans, ACC2 is the predominant isoform in both lipogenic and oxidative tissues (1,2). Phosphorylation by AMPK at Ser79 or by PKA at Ser1200 inhibits the enzymatic activity of ACC (3). ACC is a potential target of anti-obesity drugs (4,5).
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PathScan is a trademark of Cell Signaling Technology, Inc.