RayBio® Phospho- NF-κB p65(S536) and Total NF-κB p65 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human, mouse and rat cell lysates. By determining phosphorylated NF-κB p65 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human, mouse and rat phospho- NF-κB p65 and total NF-κB p65. An anti-pan NF-κB p65 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and NF-κB p65 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti- NF-κB p65 (S536) antibody is used to detect phosphorylated NF-κB p65 or biotinylated anti-NF-κB p65 antibody is used to detect pan NF-κB p65. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of NF-κB p65(S536) or pan NF-κB p65 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• 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

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Anti-Phospho Antibody
• Anti-Pan Antibody (*not included in phospho-only version)
• HRP-Conjugated Secondary Antibody
• Assay Diluent
• TMB One-Step Substrate
• Stop Solution
• Lysis Buffer
• Positive Control Sample

• 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

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.

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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), 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. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.