Cell Death
MELK Colorimetric Cell-Based ELISA
- SKU:
- CBCAB01099
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Cell Death
- Reactivity:
- Human
- Detection Method:
- Colorimetric
Description
| Product Name: | MELK Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01099 |
| ELISA Type: | Cell-Based |
| Target: | MELK |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The MELK Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MELK protein expression profile in cells. The kit can be used for measuring the relative amounts of MELK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MELK.
Qualitative determination of MELK concentration is achieved by an indirect ELISA format. In essence, MELK is captured by MELK-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: 9833, UniProt ID: Q14680, OMIM: 607025, Unigene: Hs.184339 |
| Gene Symbol: | MELK |
| Sub Type: | None |
| UniProt Protein Function: | MELK: Serine/threonine-protein kinase involved in various processes such as cell cycle regulation, self-renewal of stem cells, apoptosis and splicing regulation. Has a broad substrate specificity; phosphorylates BCL2L14, CDC25B, MAP3K5/ASK1 and ZNF622. Acts as an activator of apoptosis by phosphorylating and activating MAP3K5/ASK1. Acts as a regulator of cell cycle, notably by mediating phosphorylation of CDC25B, promoting localization of CDC25B to the centrosome and the spindle poles during mitosis. Plays a key role in cell proliferation and carcinogenesis. Required for proliferation of embryonic and postnatal multipotent neural progenitors. Phosphorylates and inhibits BCL2L14, possibly leading to affect mammary carcinogenesis by mediating inhibition of the pro-apoptotic function of BCL2L14. Also involved in the inhibition of spliceosome assembly during mitosis by phosphorylating ZNF622, thereby contributing to its redirection to the nucleus. May also play a role in primitive hematopoiesis. Monomer. Interacts with ZNF622 and PPP1R8. Up-regulated in many cancers cells. Up-regulated upon treatment with radiation or 5-fluorouracil (5-FU) in colorectal cancer cells, suggesting that it might be associated with increased resistance of colorectal cells against radiation and 5- FU. Down-regulated upon siomycin A, a thiazole antibiotic, treatment, leading to inhibit tumor growth in vivo. Expressed in placenta, kidney, thymus, testis, ovary and intestine. Activated by autophosphorylation of the T-loop at Thr-167 and Ser-171: in contrast to other members of the SNF1 subfamily, phosphorylation at Thr-167 is not mediated by STK11/LKB1 but via autophosphorylation instead. Inhibited by calcium-binding. Kinase activity is also regulated by reducing agents: dithiothreitol (DTT) or reduced glutathione are required for kinase activity in vitro; such dependence is however not due to the presence of disulfide bonds. Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. SNF1 subfamily. |
| UniProt Protein Details: | Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, CAMK; EC 2.7.10.2; RNA splicing; EC 2.7.11.1; CAMK group; CAMKL family; MELK subfamily Chromosomal Location of Human Ortholog: 9p13.2 Cellular Component: cell cortex; membrane; nucleus; plasma membrane Molecular Function:calcium ion binding; non-membrane spanning protein tyrosine kinase activity; protein binding; protein serine/threonine kinase activity Biological Process: apoptosis; cell proliferation; G2/M transition of mitotic cell cycle; hemopoiesis; positive regulation of apoptosis; protein amino acid autophosphorylation |
| UniProt Code: | Q14680 |
| NCBI GenInfo Identifier: | 50400857 |
| NCBI Gene ID: | 9833 |
| NCBI Accession: | Q14680.3 |
| UniProt Secondary Accession: | Q14680,A6P3A7, A6P3A8, B1AMQ6, B7Z1E6, B7Z5M5, B7Z6Q7 B7Z6R8, B7Z6Y0, B7Z7Q1, D3DRP8, F5H0Y0, |
| UniProt Related Accession: | Q14680 |
| Molecular Weight: | 69,116 Da |
| NCBI Full Name: | Maternal embryonic leucine zipper kinase |
| NCBI Synonym Full Names: | maternal embryonic leucine zipper kinase |
| NCBI Official Symbol: | MELK |
| NCBI Official Synonym Symbols: | HPK38 |
| NCBI Protein Information: | maternal embryonic leucine zipper kinase |
| UniProt Protein Name: | Maternal embryonic leucine zipper kinase |
| UniProt Synonym Protein Names: | Protein kinase Eg3; pEg3 kinase; Protein kinase PK38; hPK38; Tyrosine-protein kinase MELK (EC:2.7.10.2) |
| Protein Family: | Maternal embryonic leucine zipper kinase |
| UniProt Gene Name: | MELK |
| UniProt Entry Name: | MELK_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-MELK 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.)
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