RayBio® Phospho- NF-κB p65(S536) 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. 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. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG 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) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
Rapidly measure phosphorylated protein in lysates
Screen numerous different cell lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material
Application Notes
Kit Components
Pre-Coated 96-well Strip Microplate
Wash Buffer
Anti-Phospho Antibody
HRP-Conjugated Secondary Antibody
Assay Diluent
TMB One-Step Substrate
Stop Solution
Lysis Buffer
Positive Control Sample
Other Materials Required
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
Protocol Outline
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.
Typical Data
Positive Control
HeLa cells were treated with TNFα and Calyculin A at 37°C for 5 min. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
Calyculin A Stimulation of HeLa Cell Line
HeLa cells were treated or untreated with TNFα and 100nM Calyculin A for 5 min. Cell lysates were analyzed using this phosphoELISA and Western Blot.
Storage/Stability
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.
Sok Kuan Wong, Kok-Yong Chin, Fairus Ahmad, Soelaiman Ima-Nirwana, Regulation of inflammatory response and oxidative stress by tocotrienol in a rat model of non-alcoholic fatty liver disease, Journal of Functional Foods, Volume 74, 2020, 104209, ISSN 1756-4646, https://doi.org/10.1016/j.jff.2020.104209
I have used this kit for microglia cells and it worked very well. It is sensitive enough to show the phosphorylation of the NF-kB signaling pathway and well-explained all steps in the protocol.
