ACC1 Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00154
€499

Description

ACC1 Cell-Based ELISA Kit

The ACC1 Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor ACC1 protein expression profile in cells. The kit can be used for measuring the relative amounts of ACC1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ACC1 .

How does our ACC1 Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of ACC1 concentration is achieved by an indirect ELISA format. In essence, ACC1 is captured by ACC1 -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

ACC1 Colorimetric Cell-Based ELISA Kit - Information

Product Name:ACC1 Colorimetric Cell-Based ELISA Kit
Product Code:/SKUCBCAB00154
Description:The ACC1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ACC1 protein expression profile in cells. The kit can be used for measuring the relative amounts of ACC1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ACC1.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Database Links:Gene ID: 31, UniProt ID: Q13085, OMIM #: 200350, Unigene #: Hs.160556
Format:96-Well Microplate
NCBI Gene Symbol:ACACA
Sub Type:None
Target Name:ACC1

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
HRP-Conjugated Anti-Rabbit IgG Antibody12 ml
HRP-Conjugated Anti-Mouse IgG Antibody12 ml
SDS Solution12 ml
Stop Solution24 ml
Ready-to-Use Substrate12 ml
Crystal Violet Solution12 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µl to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µl of 1x primary antibodies (Anti-ACC1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µl of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µl of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µl of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

ACC1 - Protein Information

UniProt Protein Function:ACC1: a subunit of acetyl-CoA carboxylase (ACC), a multifunctional enzyme system. Catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. Phosphorylation by AMPK or PKA inhibits the enzymatic activity of ACC. ACC-alpha is the predominant isoform in liver, adipocyte and mammary gland. ACC-beta is the major isoform in skeletal muscle and heart. Phosphorylation regulates its activity.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - propanoate; Carbohydrate Metabolism - pyruvate; Ligase; EC 6.4.1.2; EC 6.3.4.14; Lipid Metabolism - fatty acid biosynthesis

Chromosomal Location of Human Ortholog: 17q21

Cellular Component: actin cytoskeleton; cytoplasm; cytosol; mitochondrion; nucleolus

Molecular Function:acetyl-CoA carboxylase activity; ATP binding; biotin carboxylase activity; metal ion binding; protein binding

Biological Process: acetyl-CoA metabolic process; biotin metabolic process; carnitine shuttle; cellular lipid metabolic process; energy reserve metabolic process; fatty acid biosynthetic process; lipid homeostasis; multicellular organismal protein metabolic process; positive regulation of cellular metabolic process; protein homotetramerization; tissue homeostasis; triacylglycerol biosynthetic process; vitamin metabolic process; water-soluble vitamin metabolic process

Disease: Acetyl-coa Carboxylase Deficiency

NCBI Summary:Acetyl-CoA carboxylase (ACC) is a complex multifunctional enzyme system. ACC is a biotin-containing enzyme which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. There are two ACC forms, alpha and beta, encoded by two different genes. ACC-alpha is highly enriched in lipogenic tissues. The enzyme is under long term control at the transcriptional and translational levels and under short term regulation by the phosphorylation/dephosphorylation of targeted serine residues and by allosteric transformation by citrate or palmitoyl-CoA. Multiple alternatively spliced transcript variants divergent in the 5' sequence and encoding distinct isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q13085
NCBI GenInfo Identifier:118601083
NCBI Gene ID:31
NCBI Accession:Q13085.2
UniProt Secondary Accession:Q13085,Q6KEV6, Q6XDA8, Q7Z2G8, Q7Z561, Q7Z563, Q7Z564 Q86WB2, Q86WB3, B2RP68,
UniProt Related Accession:Q13085
Molecular Weight:269,999 Da
NCBI Full Name:Acetyl-CoA carboxylase 1
NCBI Synonym Full Names:acetyl-CoA carboxylase alpha
NCBI Official Symbol:ACACA  
NCBI Official Synonym Symbols:ACC; ACAC; ACC1; ACCA; ACACAD  
NCBI Protein Information:acetyl-CoA carboxylase 1
UniProt Protein Name:Acetyl-CoA carboxylase 1
UniProt Synonym Protein Names:ACC-alphaIncluding the following 1 domains:Biotin carboxylase (EC:6.3.4.14
UniProt Gene Name:ACACA  
UniProt Entry Name:ACACA_HUMAN
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