NF-kB p65 ELISA Kit

Catalog No. ABIN8070288

This Colorimetric ELISA kit is designed for the quantitative measurement of Human, Rat, Rabbit, Dog and Frog NF-kB p65.

Quick Overview for NF-kB p65 ELISA Kit (ABIN8070288)

Target: NF-kB p65 (NFkBP65) (Nuclear Factor-kB p65 (NFkBP65))

Binding Specificity: S-nitrosylated

Reactivity: Human, Rat, Rabbit, Dog, Frog

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Cell Lysate, Tissue Lysate

Product Details NF-kB p65 ELISA Kit

Purpose: S-Nitrosylated NFkB ELISA Kit uses a modified 'biotin-switch' method to allow for the direct visualization of human, rat, canine (dog), frog, and rabbit S-Nitrosylated NFkB by ELISA application.

Analytical Method: Quantitative

Components:

Size / Qty
• Anti-Pan-NFKBp65 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 NF-kB p65

Target: NF-kB p65 (NFkBP65) (Nuclear Factor-kB p65 (NFkBP65))

Alternative Name: NF-kB P65

Background:

Gene Symbols: RELA, NFKB3

Background: The S-Nitrosylated NF-KB p65 ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human and mouse S-Nitrosylated NF-KB p65 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 NF-KB p65 antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and NF-KB p65 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 NF-KB p65. 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 NF-KB p65 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): Transcription factor p65 (Nuclear factor NF-kappa-B p65 subunit) (Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3)

Gene ID: 5970

UniProt: Q04206

Pathways: NF-kappaB Signaling, RTK Signaling, TCR Signaling, TLR Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Hepatitis C, Toll-Like Receptors Cascades, S100 Proteins