Hexokinase-3 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01036
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Detection Method:
Colorimetric
Frequently bought together:

Description

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Hexokinase-3 Colorimetric Cell-Based ELISA

The Hexokinase-3 Colorimetric Cell-Based ELISA Kit from AssayGenie is a powerful tool for measuring the activity of hexokinase-3 in cell samples. This kit offers high sensitivity and specificity, allowing for accurate and reproducible results in a variety of research applications.Hexokinase-3 is an important enzyme involved in glucose metabolism, playing a crucial role in regulating cellular energy production. Dysregulation of hexokinase-3 has been linked to various diseases such as cancer, diabetes, and neurodegenerative disorders, making it a key target for research and drug development.

With the Hexokinase-3 Colorimetric Cell-Based ELISA Kit, researchers can easily quantify hexokinase-3 activity in cell samples, providing valuable insights into the role of this enzyme in disease pathology and potential therapeutic interventions. Trust AssayGenie for reliable and accurate results in your hexokinase-3 research.

Product Name:Hexokinase-3 Colorimetric Cell-Based ELISA
Product Code:CBCAB01036
ELISA Type:Cell-Based
Target:Hexokinase-3
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Hexokinase-3 concentration is achieved by an indirect ELISA format. In essence, Hexokinase-3 is captured by Hexokinase-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: 3101, UniProt ID: P52790, OMIM: 142570, Unigene: Hs.411695
Gene Symbol:HK3
Sub Type:None
UniProt Protein Function:HK3: a glycolytic enzyme that catalyzes the reaction ATP + D-hexose = ADP + D-hexose 6-phosphate. The first and rate-limiting step in glycosis, a pathway that produces energy in the form of ATP from glucose. An allosteric enzyme inhibited by its product glucose-6-phosphate (Glc-6-P). Acts as a ""glucose sensor"" by trapping glucose inside the cell by catalyzing its phosphorylation to produce Glc-6-P. In vertebrates there are four major glucose-phosphorylating isoenzymes, designated hexokinase I, II, III and IV (glucokinase).
UniProt Protein Details:

Protein type:Transferase; Carbohydrate Metabolism - amino sugar and nucleotide sugar; Enzyme, cellular metabolism; Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - fructose and mannose; EC 2.7.1.1; Kinase, other; Carbohydrate Metabolism - starch and sucrose; Carbohydrate Metabolism - galactose

Chromosomal Location of Human Ortholog: 5q35.2

Cellular Component: cytosol

Molecular Function:fructokinase activity; glucokinase activity; hexokinase activity; mannokinase activity

Biological Process: cell glucose homeostasis; glucose transport; glycolysis

NCBI Summary:Hexokinases phosphorylate glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways. This gene encodes hexokinase 3. Similar to hexokinases 1 and 2, this allosteric enzyme is inhibited by its product glucose-6-phosphate. [provided by RefSeq, Apr 2009]
UniProt Code:P52790
NCBI GenInfo Identifier:206729871
NCBI Gene ID:3101
NCBI Accession:P52790.2
UniProt Secondary Accession:P52790,Q8N1E7,
UniProt Related Accession:P52790
Molecular Weight:99,025 Da
NCBI Full Name:Hexokinase-3
NCBI Synonym Full Names:hexokinase 3
NCBI Official Symbol:HK3  
NCBI Official Synonym Symbols:HXK3; HKIII  
NCBI Protein Information:hexokinase-3
UniProt Protein Name:Hexokinase-3
UniProt Synonym Protein Names:Hexokinase type III; HK III
Protein Family:Probable histidine kinase
UniProt Gene Name:HK3  
UniProt Entry Name:HXK3_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-Hexokinase-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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