GCK ELISA Kit

Catalog No. ABIN8068924

The Human GCK ELISA Kit (ABIN8068924) is a Colorimetric ELISA Kit designed to quantify Human GCK.

Quick Overview for GCK ELISA Kit (ABIN8068924)

Target: GCK (Glucokinase (Hexokinase 4) (GCK))

Binding Specificity: S-nitrosylated, total

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Cell Lysate, Tissue Lysate

Product Details GCK ELISA Kit

Purpose: Measures s-nitrosylated Glucokinase (GCK) and total Glucokinase in human cell and tissue lysates, enabling precise assessment of Glucokinase s-nitrosylation by ELISA. This assay can be developed for other species (mouse, rat, etc.) upon request. Contact techsupport@raybiotech.com.

Analytical Method: Quantitative

Components:

Size / Qty
• Microplate Coated With Anti-Pan-Glucokinase Antibody
• Positive Control
• HRP-Conjugated Streptavidin
• 200 μL
• Wash Buffer
• Assay Diluent
• Lysis Buffer
• TMB One-Step Substrate Reagent
• Stop Solution
• S-Nitrosylation Buffer A
• S-Nitrosylation Buffer B
• S-Nitrosylation Buffer C
• S-Nitrosylation Buffer D
• S-Nitrosylation Buffer E
• S-Nitrosylation Blocking Reagent
• S-Nitrosylation Reduction Reagent I
• S-Nitrosylation Reduction Reagent II
• S-Nitrosylation Labeling Reagent

Material not included:

• Acetone (pre-chilled (-20 °C))
• Microplate reader capable of measuring absorbance at 450 nm
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Log-log graph paper or computer and software for ELISA data analysis
• Absorbent paper
• Tubes to prepare positive control or sample dilutions
• Distilled or deionized water
• Shaker

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and Positive Control as instructed.
2. Block unmodified free cysteines using prepared S-Nitrosylation Blocking Buffer at 50 °C with gentle rocking.
3. Precipitate proteins with ice-cold acetone.
4. Wash protein pellet twice with cold acetone/water mixture.
5. Resuspend protein pellets in Buffers and adding Reducing and Labeling Reagents.
6. Precipitate proteins with ice-cold acetone.
7. Resuspend protein pellets.
8. Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.
9. Add 100 μL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
10. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
11. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

Restrictions: For Research Use only

Handling

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.

Target Details for GCK

Target: GCK (Glucokinase (Hexokinase 4) (GCK))

Alternative Name: Glucokinase

Background:

Gene Symbols: GCK

Background: The S-Nitrosylated-Glucokinase ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human S-Nitrosylated Glucokinase by ELISA. In this S-Nitrosylation biotin switch assay, unmodified free cysteines are first blocked. S-Nitrosylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was S-Nitrosylated. Biotinylation of the newly formed thiol groups can then be detected by ELISA. An anti-pan Glucokinase antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and Glucokinase present in a sample is bound to the wells by the immobilized antibody. The wells are washed and HRP-conjugated streptavidin is used to detect S-Nitrosylated Glucokinase. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of S-Nitrosylated Glucokinase bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Protein Name (Synonyms): Hexokinase-4 (HK4), Glucokinase, Hexokinase type IV (HK IV), Hexokinase-D

Gene ID: 2645

UniProt: P35557

Pathways: MAPK Signaling, Positive Regulation of Peptide Hormone Secretion, Carbohydrate Homeostasis, Cellular Glucan Metabolic Process, Regulation of Carbohydrate Metabolic Process