Description
QORX Colorimetric Cell-Based ELISA
The QORX Colorimetric Cell-Based ELISA Kit is a powerful tool for analyzing angiogenin levels in various biological samples. This kit is specifically designed for accurate measurement of angiogenin in serum, plasma, and cell culture supernatants, offering high sensitivity and specificity for reliable and reproducible results.Angiogenin is a key protein involved in angiogenesis, a process crucial for blood vessel formation and cell proliferation. Dysregulation of angiogenin has been linked to a variety of diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions.
As such, the QORX Colorimetric Cell-Based ELISA Kit serves as an invaluable resource for researchers studying these diseases and exploring potential therapeutic interventions.With easy-to-use protocols and a user-friendly format, the QORX Colorimetric Cell-Based ELISA Kit is an essential tool for any laboratory conducting angiogenin research. Trust in the accuracy and precision of this kit to support your investigations and advance the understanding of angiogenin's role in health and disease.
| Product Name: | QORX Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01129 |
| ELISA Type: | Cell-Based |
| Target: | QORX |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The QORX Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect QORX protein expression profile in cells. The kit can be used for measuring the relative amounts of QORX in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on QORX.
Qualitative determination of QORX concentration is achieved by an indirect ELISA format. In essence, QORX is captured by QORX-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: 9540, UniProt ID: Q53FA7, OMIM: 605171, Unigene: Hs.50649 |
| Gene Symbol: | QORX |
| Sub Type: | None |
| UniProt Protein Function: | TP53I3: May be involved in the generation of reactive oxygen species (ROS). Has low NADPH-dependent beta-naphthoquinone reductase activity, with a preference for 1,2-beta-naphthoquinone over 1,4-beta-naphthoquinone. Has low NADPH-dependent diamine reductase activity (in vitro). Belongs to the zinc-containing alcohol dehydrogenase family. Quinone oxidoreductase subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:EC 1.-.-.-; Oxidoreductase Chromosomal Location of Human Ortholog: 2p23.3 Molecular Function:NADPH:quinone reductase activity; protein homodimerization activity; quinone binding; zinc ion binding Biological Process: NADP metabolic process |
| NCBI Summary: | The protein encoded by this gene is similar to oxidoreductases, which are enzymes involved in cellular responses to oxidative stresses and irradiation. This gene is induced by the tumor suppressor p53 and is thought to be involved in p53-mediated cell death. It contains a p53 consensus binding site in its promoter region and a downstream pentanucleotide microsatellite sequence. P53 has been shown to transcriptionally activate this gene by interacting with the downstream pentanucleotide microsatellite sequence. The microsatellite is polymorphic, with a varying number of pentanucleotide repeats directly correlated with the extent of transcriptional activation by p53. It has been suggested that the microsatellite polymorphism may be associated with differential susceptibility to cancer. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2011] |
| UniProt Code: | Q53FA7 |
| NCBI GenInfo Identifier: | 76789665 |
| NCBI Gene ID: | 9540 |
| NCBI Accession: | Q53FA7.2 |
| UniProt Secondary Accession: | Q53FA7,O14679, O14685, Q38G78, Q6JLE7, Q9BWB8, D6W533 |
| UniProt Related Accession: | Q53FA7 |
| Molecular Weight: | 25,402 Da |
| NCBI Full Name: | Quinone oxidoreductase PIG3 |
| NCBI Synonym Full Names: | tumor protein p53 inducible protein 3 |
| NCBI Official Symbol: | TP53I3 |
| NCBI Official Synonym Symbols: | PIG3 |
| NCBI Protein Information: | quinone oxidoreductase PIG3 |
| UniProt Protein Name: | Quinone oxidoreductase PIG3 |
| UniProt Synonym Protein Names: | Tumor protein p53-inducible protein 3; p53-induced gene 3 protein |
| Protein Family: | Quinone oxidoreductase |
| UniProt Gene Name: | TP53I3 |
| UniProt Entry Name: | QORX_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-QORX 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.)
