GAPDH ELISA Kit

Catalog No. ABIN8068969

The Human, Mouse and Rat GAPDH ELISA Kit (ABIN8068969) is a Colorimetric ELISA Kit designed to quantify Human, Mouse and Rat GAPDH.

Quick Overview for GAPDH ELISA Kit (ABIN8068969)

Target: GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH))

Binding Specificity: S-nitrosylated

Reactivity: Human, Mouse, Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Cell Lysate, Tissue Lysate

Product Details GAPDH ELISA Kit

Purpose: S-Nitrosylated GAPDH ELISA Kit uses a modified 'biotin-switch' method to allow for the direct visualization of human, rat, and mouse S-Nitrosylated GAPDH by ELISA application.

Analytical Method: Quantitative

Components:

Size / Qty
• Anti-Pan-GAPDH Microplate
• 96 wells
• Positive Control
• HRP-Streptavidin
• 200 μL
• Wash Buffer
• Assay Diluent B
• 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 Buffer containing 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-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 GAPDH

Target: GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH))

Alternative Name: GAPDH

Background:

Gene Symbols: GAPDH, GAPD, CDABP0047, OK/SW-cl.12

Background: The S-Nitrosylated GAPDH ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human and mouse S-Nitrosylated GAPDH 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 GAPDH antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and GAPDH 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 GAPDH. 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 GAPDH 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): Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) (Peptidyl-cysteine S-nitrosylase GAPDH) (EC 2.6.99.-)

Gene ID: 2597

UniProt: P04406