Description
NYREN18 Colorimetric Cell-Based ELISA Kit
The NYREN18 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the precise measurement of angiogenin levels in cell culture samples. This innovative kit offers exceptional sensitivity and specificity, guaranteeing accurate and consistent results for a variety of research purposes.Angiogenin is a key protein in the process of angiogenesis, playing a vital role in the formation of blood vessels and regulating cell growth. Its implications in conditions like cancer, cardiovascular diseases, and neurodegenerative disorders underscore its importance as a biomarker for investigating these diseases and exploring therapeutic interventions.
With the NYREN18 Colorimetric Cell-Based ELISA Kit, researchers can delve deeper into the role of angiogenin in disease pathogenesis and treatment, paving the way for novel discoveries and advancements in biomedical research. Experience the precision and reliability of this state-of-the-art kit for your research needs.
| Product Name: | NYREN18 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00786 |
| ELISA Type: | Cell-Based |
| Target: | NYREN18 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The NYREN18 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NYREN18 protein expression profile in cells. The kit can be used for measuring the relative amounts of NYREN18 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NYREN18.
Qualitative determination of NYREN18 concentration is achieved by an indirect ELISA format. In essence, NYREN18 is captured by NYREN18-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: 51667, UniProt ID: Q9Y5A7, OMIM: 607981, Unigene: Hs.647082/Hs.662237 |
| Gene Symbol: | NUB1 |
| Sub Type: | None |
| UniProt Protein Function: | NYREN18: Specific down-regulator of the NEDD8 conjugation system. Recruits NEDD8, UBD, and their conjugates to the proteasome for degradation. Isoform 1 promotes the degradation of NEDD8 more efficiently than isoform 2. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Ubiquitin conjugating system Chromosomal Location of Human Ortholog: 7q36 Molecular Function:protein binding Biological Process: positive regulation of proteasomal ubiquitin-dependent protein catabolic process; protein ubiquitination; ubiquitin-dependent protein catabolic process |
| NCBI Summary: | This gene encodes a protein that functions as a negative regulator of NEDD8, a ubiquitin-like protein that conjugates with cullin family members in order to regulate vital biological events. The protein encoded by this gene regulates the NEDD8 conjugation system post-transcriptionally by recruiting NEDD8 and its conjugates to the proteasome for degradation. This protein interacts with the product of the AIPL1 gene, which is associated with Leber congenital amaurosis, an inherited retinopathy, and mutations in that gene can abolish interaction with this protein, which may contribute to the pathogenesis. This protein is also known to accumulate in Lewy bodies in Parkinson's disease and dementia with Lewy bodies, and in glial cytoplasmic inclusions in multiple system atrophy, with this abnormal accumulation being specific to alpha-synucleinopathy lesions. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Aug 2011] |
| UniProt Code: | Q9Y5A7 |
| NCBI GenInfo Identifier: | 37154888 |
| NCBI Gene ID: | 51667 |
| NCBI Accession: | Q9Y5A7.2 |
| UniProt Secondary Accession: | Q9Y5A7,O95422, Q75MR9, Q8IX22, Q9BXR2, |
| UniProt Related Accession: | Q9Y5A7 |
| Molecular Weight: | 69,119 Da |
| NCBI Full Name: | NEDD8 ultimate buster 1 |
| NCBI Synonym Full Names: | negative regulator of ubiquitin like proteins 1 |
| NCBI Official Symbol: | NUB1 |
| NCBI Official Synonym Symbols: | BS4; NUB1L; NYREN18 |
| NCBI Protein Information: | NEDD8 ultimate buster 1 |
| UniProt Protein Name: | NEDD8 ultimate buster 1 |
| UniProt Synonym Protein Names: | Negative regulator of ubiquitin-like proteins 1; Renal carcinoma antigen NY-REN-18 |
| Protein Family: | NEDD8 ultimate buster |
| UniProt Gene Name: | NUB1 |
| UniProt Entry Name: | NUB1_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-NYREN18 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.)
