Figure 1. The PathScan® Total RIP3 Sandwich ELISA Kit (Human Preferred) #81714 detects human RIP3 protein in Colo 205 cells, but cannot detect mouse RIP3 protein expressed in L-929 cells. The relationship between lysate protein concentration from Colo 205 or L-929 cells and the absorbance at 450 nm is shown. Unstarved Colo 205 or L-929 cells were harvested and then lysed.
The PathScan® Total RIP3 Sandwich ELISA Kit (Human Preferred) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of RIP3 protein. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with RIP3 protein in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of RIP3 protein. Antibodies in this kit are custom formulations specific to kit.
NOTE: This protocol is for PathScan® kits that use an HRP directly conjugated to the detection antibody (1-step method), rather than a 2-step method where the detection antibody and a secondary-HRP are added sequentially.
Refer to product-specific datasheets for assay incubation temperature.
NOTE: Prepare solutions with deionized/purified water or equivalent.
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
created July 2020
Protocol Id: 2144
The PathScan® Total RIP3 Sandwich ELISA Kit (Human Preferred) detects endogenous levels of RIP3 protein. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The receptor-interacting protein (RIP) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that trigger pro-survival and inflammatory responses through the activation of NF-κB, as well as pro-apoptotic pathways (1). In addition to the kinase domain, RIP contains a death domain responsible for interaction with the death domain receptor Fas and recruitment to TNF-R1 through interaction with TRADD (2,3). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (4,5). RIP also interacts with TNF-receptor-associated factors (TRAFs) and can recruit IKKs to the TNF-R1 signaling complex via interaction with NEMO, leading to IκB phosphorylation and degradation (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (2,3). Caspase-8-dependent cleavage of the RIP death domain can trigger the apoptotic activity of RIP (8).
Receptor-interacting protein 3 (RIP3) was originally found to interact with RIP and the TNF receptor complex to induce apoptosis and activation of NF-κB (9,10). It has subsequently been shown that the association between RIP and RIP3 is a key component of a signaling pathway that results in programmed necrosis (necroptosis), a necrotic-like cell death induced by TNF in the presence of caspase inhibitors (11-13). RIP3 is phosphorylated at Ser227 and targets the phosphorylation of mixed lineage kinase domain-like protein (MLKL), which is critical for necroptosis (14).
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