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PathScan® Phospho-Acetyl-CoA Carboxylase (Ser79) Sandwich ELISA Kit #7986
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.Learn more about how we get our images
Gallery: PathScan® Phospho-Acetyl-CoA Carboxylase (Ser79) Sandwich ELISA Kit #7986
- 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.
- 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® Phospho-Acetyl-CoA Carboxylase (Ser79) Sandwich ELISA Kit is shown.
|Product Includes||Volume||Solution Color|
|Phospho-ACC (Ser79) Ab Coated Microwells||96 assays|
|ACC Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml||Yellowish|
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
- Bring all microwell strips to room temperature before use.
- Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
1X Cell Lysis Buffer: PathScan® Sandwich ELISA Lysis Buffer (#7018) 1X: This buffer is ready to use as is. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
- TMB Substrate: (#7004).
- STOP Solution: (#7002).
B. Preparing Cell Lysates
For adherent cells
- Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
- Remove media and rinse cells once with ice-cold 1X PBS.
- Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
- Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
- Sonicate lysates on ice.
- Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.
For suspension cells
- Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
- Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
- Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
- Sonicate lysates on ice.
C. Test Procedure
- After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately.
- Cell lysates can be undiluted or diluted with sample diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
- Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.
- Gently remove the tape and wash wells:
- Discard plate contents into a receptacle.
- Wash 4 times with 1X wash buffer, 200 µl each time per well.
- For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
- Clean the underside of all wells with a lint-free tissue.
- Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr.
- Repeat wash procedure (Section C, Step 4).
- Add 100 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 min at 37°C.
- Repeat wash procedure (Section C, Step 4).
- Add 100 µl of TMB substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
- Read results
- Visual Determination: Read within 30 min after adding STOP solution.
- Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.
posted June 2005
revised November 2013
protocol id: 208
The PathScan® Phospho-Acetyl-CoA Carboxylase (Ser79) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetyl-CoA carboxylase (ACC) when phosphorylated at Ser79. A phospho-ACC (Ser79) rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-ACC protein is captured by the coated antibody. Following extensive washing, an ACC mouse detection mAb is added to detect the captured ACC protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of ACC phosphorylated at Ser79.
Antibodies in kit are custom formulations specific to kit.
PathScan® Phospho-Acetyl-CoA Carboxylase (Ser79) Sandwich ELISA Kit detects endogenous levels of ACC protein when phosphorylated at Ser79 as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity: Human
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. PathScan® is a trademark of Cell Signaling Technology, Inc.