Description
p44/42 MAP Kinase (Phospho-Tyr204)Colorimetric Cell-Based ELISA Kit
The p44/42 MAP Kinase Phospho-Tyr204 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting and quantifying levels of phosphorylated p44/42 MAP kinase in cell lysates. This kit provides high sensitivity and specificity, allowing for accurate and reliable results in a variety of research applications.p44/42 MAP kinase, also known as ERK1/2, plays a crucial role in cell signaling pathways involved in cell growth, proliferation, and differentiation. Phosphorylation of Tyr204 is a key regulatory event in the activation of p44/42 MAP kinase, making it a valuable marker for studying signaling pathways involved in various physiological and pathological processes.
By using the p44/42 MAP Kinase Phospho-Tyr204 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the activation status of p44/42 MAP kinase in response to various stimuli, enabling a better understanding of cellular signaling events and potential therapeutic targets.
| Product Name: | p44/42 MAP Kinase (Phospho-Tyr204)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01523 |
| ELISA Type: | Cell-Based |
| Target: | p44/42 MAP Kinase (Phospho-Tyr204) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The p44/42 MAP Kinase (Phospho-Tyr204) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p44/42 MAP Kinase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p44/42 MAP Kinase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p44/42 MAP Kinase phosphorylation.
Qualitative determination of p44/42 MAP Kinase (Phospho-Tyr204) concentration is achieved by an indirect ELISA format. In essence, p44/42 MAP Kinase (Phospho-Tyr204) is captured by p44/42 MAP Kinase (Phospho-Tyr204)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 5595/5594, UniProt ID: P27361/P28482, OMIM: 601795/176948, Unigene: Hs.861/Hs.431850 |
| Gene Symbol: | MAPK1/MAPK3 |
| Sub Type: | Phospho |
| UniProt Protein Function: | ERK1: a serine/threonine kinase of the GMGC group that plays a critical role in the regulation of cell growth and differentiation. ERK1 (MAPK3) and ERK2 (MAPK1) play central roles in MAPK cascades and are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Depending on the cellular context, MAPK cascades mediate diverse biological functions such as cell growth, adhesion, survival and differentiation through the regulation of transcription, translation, cytoskeletal rearrangements. MAPK cascades also plays a role in initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine residues at the sequence T*EY* by upstream MAP kinase kinases, MEK1 and -2. Phosphorylation of both the threonine and tyrosine are required for activity. This phosphorylation causes dramatic conformational changes, which enable full activation and interaction of MAPK1/ERK2 with its substrates. |
| UniProt Protein Details: | Protein type:EC 2.7.11.24; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; CMGC group; MAPK family; ERK subfamily; MAPK/ERK subfamily Chromosomal Location of Human Ortholog: 16p11.2 Cellular Component: microtubule cytoskeleton; nucleoplasm; Golgi apparatus; focal adhesion; cytoskeleton; mitochondrion; late endosome; early endosome; nuclear envelope; caveola; pseudopodium; nucleus; cytosol Molecular Function:MAP kinase activity; protein binding; phosphotyrosine binding; ATP binding; phosphatase binding Biological Process: axon guidance; DNA damage induced protein phosphorylation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; positive regulation of histone phosphorylation; viral reproduction; apoptosis; activation of MAPK activity; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; sensory perception of pain; protein amino acid phosphorylation; toll-like receptor 10 signaling pathway; BMP signaling pathway; toll-like receptor 5 signaling pathway; response to exogenous dsRNA; regulation of transcription factor activity; small GTPase mediated signal transduction; lipopolysaccharide-mediated signaling pathway; toll-like receptor 4 signaling pathway; epidermal growth factor receptor signaling pathway; platelet activation; fibroblast growth factor receptor signaling pathway; cytokine and chemokine mediated signaling pathway; MyD88-independent toll-like receptor signaling pathway; MAPKKK cascade; transcription from RNA polymerase I promoter; cell cycle; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; regulation of stress-activated MAPK cascade; organ morphogenesis; cartilage development; Ras protein signal transduction; toll-like receptor signaling pathway; insulin receptor signaling pathway; innate immune response; gene expression; positive regulation of histone acetylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of protein amino acid phosphorylation; toll-like receptor 9 signaling pathway; transcription initiation from RNA polymerase I promoter; vascular endothelial growth factor receptor signaling pathway; blood coagulation; phosphorylation; regulation of cytoskeleton organization and biogenesis |
| NCBI Summary: | The protein encoded by this gene is a member of the MAP kinase family. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act in a signaling cascade that regulates various cellular processes such as proliferation, differentiation, and cell cycle progression in response to a variety of extracellular signals. This kinase is activated by upstream kinases, resulting in its translocation to the nucleus where it phosphorylates nuclear targets. Alternatively spliced transcript variants encoding different protein isoforms have been described. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P27361 |
| NCBI GenInfo Identifier: | 232066 |
| NCBI Gene ID: | 5595 |
| NCBI Accession: | P27361.4 |
| UniProt Secondary Accession: | P27361,Q8NHX1, A8CZ58, B0LPG3, |
| UniProt Related Accession: | P27361 |
| Molecular Weight: | 379 |
| NCBI Full Name: | Mitogen-activated protein kinase 3 |
| NCBI Synonym Full Names: | mitogen-activated protein kinase 3 |
| NCBI Official Symbol: | MAPK3 |
| NCBI Official Synonym Symbols: | ERK1; ERT2; ERK-1; PRKM3; P44ERK1; P44MAPK; HS44KDAP; HUMKER1A; p44-ERK1; p44-MAPK |
| NCBI Protein Information: | mitogen-activated protein kinase 3; MAPK 1; MAP kinase isoform p44; insulin-stimulated MAP2 kinase; extracellular signal-related kinase 1; extracellular signal-regulated kinase 1; microtubule-associated protein 2 kinase |
| UniProt Protein Name: | Mitogen-activated protein kinase 3 |
| UniProt Synonym Protein Names: | ERT2; Extracellular signal-regulated kinase 1; ERK-1; Insulin-stimulated MAP2 kinase; MAP kinase isoform p44; p44-MAPK; Microtubule-associated protein 2 kinase; p44-ERK1 |
| UniProt Gene Name: | MAPK3 |
| UniProt Entry Name: | MK03_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-p44/42 MAP Kinase (Phospho-Tyr204) Antibody, Anti-p44/42 MAP Kinase 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.)
