Description
RIPK2 (Phospho-Ser176)Colorimetric Cell-Based ELISA Kit
The RIPK2 Phospho-Ser176 Colorimetric Cell-Based ELISA Kit is a highly sensitive and specific assay designed for the detection of phosphorylated RIPK2 levels in cell lysates. This kit enables accurate and reproducible results, making it suitable for a variety of research applications.RIPK2, also known as receptor-interacting serine/threonine-protein kinase 2, is a key signaling protein involved in cell death and inflammation pathways. Phosphorylation of RIPK2 at Ser176 plays a crucial role in its activation and downstream signaling effects.
Dysregulation of RIPK2 signaling has been implicated in various diseases, including inflammatory disorders and certain cancers.By detecting the phosphorylation status of RIPK2 at Ser176, researchers can better understand the molecular mechanisms underlying disease pathology and potentially identify novel therapeutic targets. The RIPK2 Phospho-Ser176 Colorimetric Cell-Based ELISA Kit is a valuable tool for studying RIPK2 signaling pathways and developing potential therapies for related diseases.
| Product Name: | RIPK2 (Phospho-Ser176)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00151 |
| ELISA Type: | Cell-Based |
| Target: | RIPK2 (Phospho-Ser176) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The RIPK2 (Phospho-Ser176) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect RIPK2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated RIPK2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on RIPK2 phosphorylation.
Qualitative determination of RIPK2 (Phospho-Ser176) concentration is achieved by an indirect ELISA format. In essence, RIPK2 (Phospho-Ser176) is captured by RIPK2 (Phospho-Ser176)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 8767, UniProt ID: O43353, OMIM: 603455, Unigene: Hs.103755 |
| Gene Symbol: | RIPK2 |
| Sub Type: | Phospho |
| UniProt Protein Function: | RIPK2: a tyrosine kinase-like kinase of the RIPK family. Activates pro-caspase-1 and pro-caspase-8. Potentiates casp-8-mediated apoptosis. May activate NF-kappaB. |
| UniProt Protein Details: | Protein type:EC 2.7.11.1; Kinase, protein; EC 2.7.10.2; Protein kinase, Ser/Thr (non-receptor); Protein kinase, TKL; TKL group; RIPK family Chromosomal Location of Human Ortholog: 8q21 Cellular Component: cytoplasm; cytoskeleton; cytosol; protein complex; vesicle Molecular Function:ATP binding; CARD domain binding; LIM domain binding; non-membrane spanning protein tyrosine kinase activity; protein binding; protein homodimerization activity; protein serine/threonine kinase activity; receptor binding; signal transducer activity Biological Process: activation of MAPK activity; activation of NF-kappaB transcription factor; adaptive immune response; apoptosis; defense response to Gram-positive bacterium; I-kappaB kinase/NF-kappaB cascade; inflammatory response; innate immune response; JNK cascade; lipopolysaccharide-mediated signaling pathway; MyD88-dependent toll-like receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; negative regulation of apoptosis; nerve growth factor receptor signaling pathway; peptidyl-tyrosine phosphorylation; positive regulation of alpha-beta T cell proliferation; positive regulation of apoptosis; positive regulation of chemokine production; positive regulation of cytokine and chemokine mediated signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of immature T cell proliferation; positive regulation of interferon-alpha production; positive regulation of interferon-beta production; positive regulation of interferon-gamma production; positive regulation of interleukin-12 production; positive regulation of interleukin-2 production; positive regulation of interleukin-6 production; positive regulation of JNK cascade; positive regulation of peptidyl-serine phosphorylation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein ubiquitination; positive regulation of T-helper 1 cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tumor necrosis factor production; response to exogenous dsRNA; signal transduction; stress-activated MAPK cascade; T cell proliferation; T cell receptor signaling pathway; toll-like receptor 10 signaling pathway; toll-like receptor 2 signaling pathway; toll-like receptor 3 signaling pathway; toll-like receptor 4 signaling pathway; toll-like receptor 5 signaling pathway; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway |
| NCBI Summary: | This gene encodes a member of the receptor-interacting protein (RIP) family of serine/threonine protein kinases. The encoded protein contains a C-terminal caspase activation and recruitment domain (CARD), and is a component of signaling complexes in both the innate and adaptive immune pathways. It is a potent activator of NF-kappaB and inducer of apoptosis in response to various stimuli. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O43353 |
| NCBI GenInfo Identifier: | 20455217 |
| NCBI Gene ID: | 8767 |
| NCBI Accession: | O43353.2 |
| UniProt Secondary Accession: | O43353,Q6UWF0, B7Z748, |
| UniProt Related Accession: | O43353 |
| Molecular Weight: | 45,582 Da |
| NCBI Full Name: | Receptor-interacting serine/threonine-protein kinase 2 |
| NCBI Synonym Full Names: | receptor interacting serine/threonine kinase 2 |
| NCBI Official Symbol: | RIPK2 |
| NCBI Official Synonym Symbols: | CCK; RICK; RIP2; CARD3; GIG30; CARDIAK |
| NCBI Protein Information: | receptor-interacting serine/threonine-protein kinase 2 |
| UniProt Protein Name: | Receptor-interacting serine/threonine-protein kinase 2 |
| UniProt Synonym Protein Names: | CARD-containing interleukin-1 beta-converting enzyme-associated kinase; CARD-containing IL-1 beta ICE-kinase; RIP-like-interacting CLARP kinase; Receptor-interacting protein 2; RIP-2; Tyrosine-protein kinase RIPK2 (EC:2.7.10.2) |
| Protein Family: | Putative zinc metalloprotease |
| UniProt Gene Name: | RIPK2 |
| UniProt Entry Name: | RIPK2_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies Anti-RIPK2 (Phospho-Ser176) Antibody, Anti-RIPK2 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
