Figure 1. Treatment of HT-29 cells with Z-VAD followed by addition of human TNF-α and SM-164 (detailed below) stimulates phosphorylation of RIP at Ser166 compared to untreated cells. The relationship between lysate protein concentration from untreated and treated HT-29 cells and immediate light generation with chemiluminescent substrate using the PathScan® Phospho-RIP (Ser166) Chemiluminescent Sandwich ELISA Kit #88918 is shown. HT-29 cells were left untreated or treated with Z-VAD (20 μM, added 30 min prior to other compounds), human TNF-α (20 ng/ml, 7 hr), and SM-164 (100 nM, 7 hr), and then lysed.
|Product Includes||Volume||Solution Color|
|RIP Rabbit mAb Coated Microwells||96 tests|
|Phospho-RIP (Ser166) Rabbit Detection mAb||1 ea||Red (Lyophilized)|
|HRP Diluent||5.5 ml||Red|
|Luminol/Enhancer Solution||3 ml|
|Stable Peroxide Buffer||3 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-RIP (Ser166) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of RIP protein phosphorylated at Ser166. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with phospho-RIP (Ser166) protein in a single step. The plate is then extensively washed and chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of RIP protein phosphorylated at Ser166.
*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. This chemiluminescent ELISA is offered in low volume microplates. Only a total volume of 50 µL (samples and reagents) are required in each microwell.
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.
Protocol Id: 2124
The PathScan® Phospho-RIP (Ser166) Chemiluminescent Sandwich ELISA Kit detects endogenous levels of RIP protein phosphorylated at Ser166. 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).
Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury (9,10). The process is negatively regulated by caspases and is initiated through a complex containing the RIP and RIP3 kinases, typically referred to as the necrosome. Necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (11). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury (12). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (13).
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