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PRKAG1/2/3 Colorimetric Cell-Based ELISA

SKU:
CBCAB01041
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

PRKAG1/2/3 Colorimetric Cell-Based ELISA

The PRKAG1/2/3 Colorimetric Cell-Based ELISA kit is a powerful tool for detecting the activation status of the AMP-activated protein kinase (AMPK) pathway in cell-based assays. This kit offers high sensitivity and specificity, allowing for accurate and reliable measurement of PRKAG1/2/3 activation levels in a variety of cell types.The AMPK pathway plays a crucial role in cellular energy homeostasis, regulating processes such as glucose uptake, fatty acid oxidation, and mitochondrial biogenesis. Dysregulation of this pathway has been implicated in numerous diseases, including metabolic disorders, cancer, and neurodegenerative conditions.

By accurately measuring PRKAG1/2/3 activation, researchers can gain valuable insights into the underlying mechanisms of these diseases and facilitate the development of novel therapeutic strategies.Overall, the PRKAG1/2/3 Colorimetric Cell-Based ELISA kit offers a precise and efficient method for studying the AMPK pathway and its role in disease pathology. Its high sensitivity and specificity make it an invaluable tool for researchers seeking to unravel the complexities of cellular signaling pathways and identify potential targets for therapeutic intervention.

Product Name:PRKAG1/2/3 Colorimetric Cell-Based ELISA
Product Code:CBCAB01041
ELISA Type:Cell-Based
Target:PRKAG1/2/3
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The PRKAG1/2/3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PRKAG1/2/3 protein expression profile in cells. The kit can be used for measuring the relative amounts of PRKAG1/2/3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PRKAG1/2/3.

Qualitative determination of PRKAG1/2/3 concentration is achieved by an indirect ELISA format. In essence, PRKAG1/2/3 is captured by PRKAG1/2/3-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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.
Database Information:Gene ID: 5571/51422/53632/, UniProt ID: P54619/Q9UGJ0/Q9UGI9, OMIM: 602742/602743/604976, Unigene: Hs.530862/Hs.647072/Hs.591634
Gene Symbol:PRKAG1
Sub Type:None
UniProt Protein Function:AMPKG1: a non-catalytic subunit of AMPK, a highly conserved kinase of the CAMKL family. The holoenzyme consists of a catalytic subunit (alpha) and two regulatory subunits (beta, gamma). Each subunit exists as multiple genetic isoforms giving rise to 12 different possible combinations of holoenzyme. Plays a key role in regulation of energy homeostasis. Activated by an elevated AMP-ATP ratio due to environmental stress, such as heat shock, hypoxia and ischemia. AMPK phosphorylates and regulates many metabolic enzymes.
UniProt Protein Details:

Protein type:Autophagy; Protein kinase, regulatory subunit

Chromosomal Location of Human Ortholog: 12q12-q14

Cellular Component: AMP-activated protein kinase complex; cytosol; membrane; nucleoplasm

Molecular Function:ADP binding; AMP binding; AMP-activated protein kinase activity; ATP binding; cAMP-dependent protein kinase activity; cAMP-dependent protein kinase regulator activity; protein binding; protein kinase activity; protein kinase binding

Biological Process: cell cycle arrest; macroautophagy; positive regulation of protein kinase activity; protein amino acid phosphorylation; regulation of glycolysis; signal transduction; spermatogenesis

NCBI Summary:The protein encoded by this gene is a regulatory subunit of the AMP-activated protein kinase (AMPK). AMPK is a heterotrimer consisting of an alpha catalytic subunit, and non-catalytic beta and gamma subunits. AMPK is an important energy-sensing enzyme that monitors cellular energy status. In response to cellular metabolic stresses, AMPK is activated, and thus phosphorylates and inactivates acetyl-CoA carboxylase (ACC) and beta-hydroxy beta-methylglutaryl-CoA reductase (HMGCR), key enzymes involved in regulating de novo biosynthesis of fatty acid and cholesterol. This subunit is one of the gamma regulatory subunits of AMPK. Alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:P54619
NCBI GenInfo Identifier:1703037
NCBI Gene ID:5571
NCBI Accession:P54619.1
UniProt Secondary Accession:P54619,Q8N7V9, B4DDT7,
UniProt Related Accession:P54619
Molecular Weight:38,533 Da
NCBI Full Name:5'-AMP-activated protein kinase subunit gamma-1
NCBI Synonym Full Names:protein kinase AMP-activated non-catalytic subunit gamma 1
NCBI Official Symbol:PRKAG1  
NCBI Official Synonym Symbols:AMPKG  
NCBI Protein Information:5'-AMP-activated protein kinase subunit gamma-1
UniProt Protein Name:5'-AMP-activated protein kinase subunit gamma-1
Protein Family:5'-AMP-activated protein kinase
UniProt Gene Name:PRKAG1  
UniProt Entry Name:AAKG1_HUMAN
ComponentQuantity
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 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
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

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
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-PRKAG1/2/3 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.)

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