U-Blot® S-Nitrosylated NFkB p65 ELISA Kit

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.This assay enables quick and easy quantification of target proteins and allows high-throughput screening of numerous different lysates without the need for traditional Western Blot analysis. It features minimal hands-on operation time, simple workflow, and completely non-radioactive detection system, ensuring safe and convenient experimental operation.