HTRF Human Phospho-RIPK1 (Ser166) Detection Kit, 500 Assay Points

Phospho-RIPK1 (Ser166) assay principle

The Phospho-RIPK1 (Ser166) assay measures RIPK1 when phosphorylated at Ser166. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-RIPK1 (Ser166) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-RIPK1 (Ser166) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-RIPK1 (Ser166) one-plate assay protocol

Detection of Phosphorylated RIPK1 (Ser166) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Inhibition of RIPK1 kinase activity in SH-SY5Y cells using Necrostatin-1s

The human neuroblastoma cell line SH-SY5Y was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 20 minutes, before the addition of increasing doses of Necrostatin-1s. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 25 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-RIPK1 (Ser166) or Total-RIPK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Necrostatin-1s is also able to prevent the initiation of the necroptosis pathway in neuronal cells, as indicated by the inhibition of RIPK1 autophosphorylation at Ser166. In this cell line model, no modulation of Total RIPK1 was observed.

HTRF Phospho-RIPK1 (S166) assay compared to Western Blot

The human colorectal cancer cell line HT-29 was cultured in a T175 flask in complete culture medium for 48h at 37°C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 30 minutes before the simultaneous addition of SM-164 at 100 nM and human TNF-α at 100 ng/mL for 6 hours. After treatment, the cells were lyzed with 3 mL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-RIPK1 (Ser166) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF phospho-RIPK1 (Ser166) assay, 12,500 cells/well were enough to detect a significant signal, while 25,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF phospho-RIPK1 assay was 2 times more sensitive than the Western Blot technique.

RIPK1 Signaling Pathway

RIPK1 is a master regulator of cell fate-decisions by governing cell survival, apoptosis, and necroptosis pathways.

Upon binding of TNF-α to TNFR1, the receptor triggers the rapid formation of complex I at the cytoplasmic membrane by recruiting multiple proteins, including RIPK1, TRADD, TRAF2, and the cellular inhibitors of apoptosis 1 and 2 (cIAP1/2). cIAP1/2 catalyze the polyubiquitination of RIPK1, leading to the recruitment of IKKα and IKKβ and the activation of the NF-κB pro-survival pathway.

In the absence of cIAP1/2, RIPK1 is released from TNFR1 and associates with FADD and caspase 8 to form the cytosolic complex IIa, responsible for caspase 8-dependent cell apoptosis.

When caspase 8 activity is inhibited or under pathological conditions, RIPK1 interacts with RIPK3 and MLKL to form the cytosolic complex IIb (also called 'necrosome') which is involved in the initiation of necroptosis. The activation of RIPK1 by autophosphorylation at Ser166 is essential to this complex and triggers RIPK3 and MLKL activation by phosphorylation. MLKL is the most downstream effector of necroptosis: the protein oligomerizes and translocates to the plasma membrane, leading to cell membrane rupture and the release of DAMPs.