Description
MKK3 (Phospho-Thr222)Colorimetric Cell-Based ELISA Kit
The MKK3 Phospho-Thr222 Colorimetric Cell-Based ELISA Kit is designed for the accurate detection of phosphorylated MKK3 levels in cell lysates. This kit offers high sensitivity and specificity, providing researchers with reliable and reproducible results for various research applications.Phosphorylated MKK3 plays a critical role in cellular signaling pathways, including pathways involved in inflammation, stress responses, and cell proliferation.
Dysregulation of MKK3 phosphorylation has been linked to various diseases, making it an important target for therapeutic intervention and biomarker development.With easy-to-follow protocols and a straightforward assay procedure, the MKK3 Phospho-Thr222 Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers studying cellular signaling pathways and potential therapeutic targets.
| Product Name: | MKK3 (Phospho-Thr222)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01648 |
| ELISA Type: | Cell-Based |
| Target: | MKK3 (Phospho-Thr222) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The MKK3 (Phospho-Thr222) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MKK3 MAP2K3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MKK3 MAP2K3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MKK3 MAP2K3 phosphorylation.
Qualitative determination of MKK3 (Phospho-Thr222) concentration is achieved by an indirect ELISA format. In essence, MKK3 (Phospho-Thr222) is captured by MKK3 (Phospho-Thr222)-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: 5606, UniProt ID: P46734, OMIM: 602315, Unigene: Hs.514012 |
| Gene Symbol: | MAP2K3 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Dual specificity kinase. Is activated by cytokines and environmental stress in vivo. Catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in the MAP kinase p38. Part of a signaling cascade that begins with the activation of the adrenergic receptor ADRA1B and leads to the activation of MAPK14. |
| NCBI Summary: | The protein encoded by this gene is a dual specificity protein kinase that belongs to the MAP kinase kinase family. This kinase is activated by mitogenic and environmental stress, and participates in the MAP kinase-mediated signaling cascade. It phosphorylates and thus activates MAPK14/p38-MAPK. This kinase can be activated by insulin, and is necessary for the expression of glucose transporter. Expression of RAS oncogene is found to result in the accumulation of the active form of this kinase, which thus leads to the constitutive activation of MAPK14, and confers oncogenic transformation of primary cells. The inhibition of this kinase is involved in the pathogenesis of Yersina pseudotuberculosis. Multiple alternatively spliced transcript variants that encode distinct isoforms have been reported for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P46734 |
| NCBI GenInfo Identifier: | 24638466 |
| NCBI Gene ID: | 5606 |
| NCBI Accession: | P46734.2 |
| UniProt Secondary Accession: | P46734,Q99441, Q9UE71, Q9UE72, B3KSK7, |
| UniProt Related Accession: | P46734 |
| Molecular Weight: | 39,940 Da |
| NCBI Full Name: | Dual specificity mitogen-activated protein kinase kinase 3 |
| NCBI Synonym Full Names: | mitogen-activated protein kinase kinase 3 |
| NCBI Official Symbol: | MAP2K3 |
| NCBI Official Synonym Symbols: | MEK3; MKK3; MAPKK3; PRKMK3; SAPKK2; SAPKK-2 |
| NCBI Protein Information: | dual specificity mitogen-activated protein kinase kinase 3 |
| UniProt Protein Name: | Dual specificity mitogen-activated protein kinase kinase 3 |
| UniProt Synonym Protein Names: | MAPK/ERK kinase 3; MEK 3; Stress-activated protein kinase kinase 2; SAPK kinase 2; SAPKK-2; SAPKK2 |
| Protein Family: | Dual specificity mitogen-activated protein kinase kinase |
| UniProt Gene Name: | MAP2K3 |
| 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-MKK3 (Phospho-Thr222) Antibody, Anti-MKK3 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.)
