NF-kB p65 ELISA Kit

Catalog No. ABIN2748433

This Human, Rat and Mouse NF-kB p65 ELISA Kit is a Colorimetric ELISA Kit designed to quantify Human, Rat and Mouse NF-kB p65.

Quick Overview for NF-kB p65 ELISA Kit (ABIN2748433)

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

Binding Specificity: pSer536, total

Reactivity: Human, Rat, Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Cell Lysate, Tissue Lysate

Product Details NF-kB p65 ELISA Kit

Purpose: Human/Mouse/Rat Phospho-NF-KB p65 (S536) ELISA and Total NF-KB p65 Kit. This assay semi-quantitatively measures phophorylated NF-KB p65 (Ser536) and Total NF-KB p65 in lysate samples.

Analytical Method: Semi-Quantitative

Specificity: The antibody pair provided in this kit recognizes human NF-KB p65 phosphorylated at site Serine-536 and Total NF-KB p65.

Characteristics:

• Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Anti-Phospho Antibody
• Anti-Pan Antibody
• HRP-Conjugated Secondary Antibody
• Streptavidin-Conjugated HRP
• Assay Diluent
• TMB One-Step Substrate
• Stop Solution
• Lysis Buffer
• Positive Control Sample

Material not included:

• Distilled or deionized water
• 100 mL and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

Application Details

Plate: Pre-coated

Protocol:

1. Prepare all reagents and samples as instructed in the manual.
2. Add 100 μL of sample or positive control to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared primary antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared 1X HRP-Streptavidin to each well.
7. Incubate 1 h at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Assay Procedure:

Prepare all reagents and samples as instructed in the manual.
Add 100 μL of sample or positive control to each well.
Incubate 2.5 h at RT or O/N at 4 °C.
Add 100 μL of prepared primary antibody to each well.
Incubate 1 h at RT.
Add 100 μL of prepared 1X HRP-Streptavidin to each well.
Incubate 1 h at RT.
Add 100 μL of TMB One-Step Substrate Reagent to each well.
Incubate 30 min at RT.
Add 50 μL of Stop Solution to each well.
Read at 450 nm immediately.

Restrictions: For Research Use only

Handling

Storage: -20 °C

Storage Comment: Upon receipt, the kit should be stored at -20 °C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), HRP-Streptavidin (Item G), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4 °C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge and store at -20 °C. Reconstituted Positive Control (Item K) should be stored at -70 °C.

Expiry Date: 6 months

Target Details for NF-kB p65

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

Alternative Name: NFKBP65

Background: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB p65) phosphorylated at Serine-536 and total NF-KB p65.

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