Description
M3K13 Colorimetric Cell-Based ELISA
The M3K13 Colorimetric Cell-Based ELISA Kit is a powerful tool designed for the accurate detection of kinase levels in cell lysates and cell culture supernatants. This kit offers high sensitivity and specificity, providing reliable and reproducible results for use in various research applications.Kinases are essential proteins involved in cell signaling pathways, regulating various cellular processes such as growth, differentiation, and apoptosis. Dysregulation of kinase activity is associated with numerous diseases, including cancer, inflammation, and metabolic disorders, making them important targets for drug development and therapeutic interventions.
The M3K13 Colorimetric Cell-Based ELISA Kit enables researchers to study kinase activity in a quantitative and efficient manner, offering valuable insights into cell signaling pathways and potential therapeutic targets. With its user-friendly protocol and excellent performance, this kit is suitable for both basic research and drug discovery efforts in the field of cellular signaling and kinase biology.
| Product Name: | M3K13 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00932 |
| ELISA Type: | Cell-Based |
| Target: | M3K13 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The M3K13 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect M3K13 protein expression profile in cells. The kit can be used for measuring the relative amounts of M3K13 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on M3K13.
Qualitative determination of M3K13 concentration is achieved by an indirect ELISA format. In essence, M3K13 is captured by M3K13-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: 9175, UniProt ID: O43283, OMIM: 604915, Unigene: Hs.656069 |
| Gene Symbol: | MAP3K13 |
| Sub Type: | None |
| UniProt Protein Function: | LZK: a TKL kinase of the MLK family. Synergizes with antioxidant protein-1 to activate NF-kappaB. Forms a functional signaling complex with JIP-1, a scaffold protein of the c-Jun NH(2)-terminal kinase pathway. This kinase can phosphorylate and activate JNK and MKK7. Contains a dual leucine-zipper motif, and has been shown to form dimers/oligomers through its leucine-zipper motif. |
| UniProt Protein Details: | Protein type:EC 2.7.11.25; Kinase, protein; LZK subfamily; MLK family; Protein kinase, Ser/Thr (non-receptor); Protein kinase, TKL; TKL group Chromosomal Location of Human Ortholog: 3q27.2 Cellular Component: IkappaB kinase complex; membrane Molecular Function:identical protein binding; MAP kinase kinase kinase activity; protein binding; protein homodimerization activity; protein kinase binding; protein serine/threonine kinase activity Biological Process: activation of MAPKK activity; activation of NF-kappaB transcription factor; JNK cascade; protein amino acid autophosphorylation; protein amino acid phosphorylation |
| NCBI Summary: | The protein encoded by this gene is a member of serine/threonine protein kinase family. This kinase contains a dual leucine-zipper motif, and has been shown to form dimers/oligomers through its leucine-zipper motif. This kinase can phosphorylate and activate MAPK8/JNK, MAP2K7/MKK7, which suggests a role in the JNK signaling pathway. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O43283 |
| NCBI GenInfo Identifier: | 68052263 |
| NCBI Gene ID: | 9175 |
| NCBI Accession: | O43283.1 |
| UniProt Secondary Accession: | O43283,Q05BY6, Q15450, Q2NKN3, B2R6U2, B4DLE3, B4DMV2 B4DZJ4, D3DNU1, |
| UniProt Related Accession: | O43283 |
| Molecular Weight: | 108kDa |
| NCBI Full Name: | Mitogen-activated protein kinase kinase kinase 13 |
| NCBI Synonym Full Names: | mitogen-activated protein kinase kinase kinase 13 |
| NCBI Official Symbol: | MAP3K13 |
| NCBI Official Synonym Symbols: | LZK; MLK; MEKK13 |
| NCBI Protein Information: | mitogen-activated protein kinase kinase kinase 13 |
| UniProt Protein Name: | Mitogen-activated protein kinase kinase kinase 13 |
| UniProt Synonym Protein Names: | Leucine zipper-bearing kinase; Mixed lineage kinase; MLK |
| Protein Family: | Mitogen-activated protein kinase kinase kinase |
| UniProt Gene Name: | MAP3K13 |
| 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-M3K13 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.)
