Description
CERKL Colorimetric Cell-Based ELISA
The CERKl Colorimetric Cell-Based ELISA Kit is specially designed for the precise measurement of cellular responses in a high-throughput format. This innovative kit allows for the detection of specific cellular markers or proteins in a variety of cell types, providing valuable insights into cellular signaling pathways and interactions.With its high sensitivity and specificity, the CERKl Colorimetric Cell-Based ELISA Kit delivers reliable and reproducible results, making it an excellent tool for a wide range of research applications. Whether studying cell proliferation, apoptosis, or intracellular signaling, this kit offers a simple and efficient way to assess cellular responses in a cost-effective manner.
Overall, the CERKl Colorimetric Cell-Based ELISA Kit is an invaluable resource for researchers looking to gain a deeper understanding of cellular mechanisms and explore new avenues for potential therapeutic interventions. Its versatility and ease of use make it a must-have tool for any laboratory seeking to advance their research in cell biology and drug discovery.
| Product Name: | CERKL Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00387 |
| ELISA Type: | Cell-Based |
| Target: | CERKL |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The CERKL Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CERKL protein expression profile in cells. The kit can be used for measuring the relative amounts of CERKL in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CERKL.
Qualitative determination of CERKL concentration is achieved by an indirect ELISA format. In essence, CERKL is captured by CERKL-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: 375298, UniProt ID: Q49MI3, OMIM: 608380/608381, Unigene: Hs.715753 |
| Gene Symbol: | CERKL |
| Sub Type: | None |
| UniProt Protein Function: | CERKL: Has no detectable ceramide-kinase activity. Overexpression of CERKL protects cells from apoptosis in oxidative stress conditions. Defects in CERKL are the cause of retinitis pigmentosa type 26 (RP26). RP leads to degeneration of retinal photoreceptor cells. Patients typically have night vision blindness and loss of midperipheral visual field. As their condition progresses, they lose their far peripheral visual field and eventually central vision as well. RP26 inheritance is autosomal recessive. 8 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Nucleolus; Kinase, other Chromosomal Location of Human Ortholog: 2q31.3 Disease: Retinitis Pigmentosa 26 |
| NCBI Summary: | This gene was initially identified as a locus (RP26) associated with an autosomal recessive form of retinitis pigmentosa (arRP) disease. This gene encodes a protein with ceramide kinase-like domains, however, the protein does not phosphorylate ceramide and its target substrate is currently unknown. This protein may be a negative regulator of apoptosis in photoreceptor cells. Mutations in this gene cause a form of retinitis pigmentosa characterized by autosomal recessive cone and rod dystrophy (arCRD). Alternative splicing of this gene results in multiple transcript variants encoding different isoforms and non-coding transcripts.[provided by RefSeq, May 2010] |
| UniProt Code: | Q49MI3 |
| NCBI GenInfo Identifier: | 84028814 |
| NCBI Gene ID: | 375298 |
| NCBI Accession: | Q49MI3.1 |
| UniProt Secondary Accession: | Q49MI3,Q49MH9, Q49MI0, Q49MI1, Q49MI2, Q5DVJ2, Q5DVJ4 Q5DVJ5, Q6UZF6, Q6ZP59, B2RPL2, B4DEY1, |
| UniProt Related Accession: | Q49MI3 |
| Molecular Weight: | 57,632 Da |
| NCBI Full Name: | Ceramide kinase-like protein |
| NCBI Synonym Full Names: | ceramide kinase like |
| NCBI Official Symbol: | CERKL |
| NCBI Official Synonym Symbols: | RP26 |
| NCBI Protein Information: | ceramide kinase-like protein |
| UniProt Protein Name: | Ceramide kinase-like protein |
| Protein Family: | Ceramide kinase-like protein |
| UniProt Gene Name: | CERKL |
| UniProt Entry Name: | CERKL_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-CERKL 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.)
