Description
p73 (Phospho-Tyr99)Colorimetric Cell-Based ELISA Kit
The p73 Phospho-Tyr99 Colorimetric Cell-Based ELISA Kit is a powerful tool for studying the phosphorylation status of the p73 protein in a cellular context. This kit allows for the precise measurement of p73 phosphorylation at Tyr99, providing valuable insights into the regulatory mechanisms of this important protein.With its high sensitivity and specificity, this kit offers reliable and reproducible results, making it an ideal choice for researchers looking to investigate the role of p73 phosphorylation in various cellular processes.
By accurately measuring p73 phosphorylation, researchers can gain a better understanding of its function and potential implications in diseases such as cancer and neurodegenerative disorders.Overall, the p73 Phospho-Tyr99 Colorimetric Cell-Based ELISA Kit is a versatile tool for studying p73 signaling pathways and uncovering new therapeutic targets in the field of cell biology and molecular biology.
| Product Name: | p73 (Phospho-Tyr99)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01593 |
| ELISA Type: | Cell-Based |
| Target: | p73 (Phospho-Tyr99) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The p73 (Phospho-Tyr99) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p73 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p73 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p73 phosphorylation.
Qualitative determination of p73 (Phospho-Tyr99) concentration is achieved by an indirect ELISA format. In essence, p73 (Phospho-Tyr99) is captured by p73 (Phospho-Tyr99)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 7161, UniProt ID: O15350, OMIM: 601990, Unigene: Hs.697294 |
| Gene Symbol: | TP73 |
| Sub Type: | Phospho |
| UniProt Protein Function: | Participates in the apoptotic response to DNA damage. Isoforms containing the transactivation domain are pro-apoptotic, isoforms lacking the domain are anti-apoptotic and block the function of p53 and transactivating p73 isoforms. May be a tumor suppressor protein. |
| NCBI Summary: | This gene encodes a member of the p53 family of transcription factors involved in cellular responses to stress and development. It maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and thought to contain multiple tumor suppressor genes. The demonstration that this gene is monoallelically expressed (likely from the maternal allele), supports the notion that it is a candidate gene for neuroblastoma. Many transcript variants resulting from alternative splicing and/or use of alternate promoters have been found for this gene, but the biological validity and the full-length nature of some variants have not been determined. [provided by RefSeq, Feb 2011] |
| UniProt Code: | O15350 |
| NCBI GenInfo Identifier: | 8928244 |
| NCBI Gene ID: | 7161 |
| NCBI Accession: | O15350.1 |
| UniProt Secondary Accession: | O15350,O15351, Q17RN8, Q5TBV5, Q5TBV6, Q8NHW9, Q8TDY5 Q8TDY6, B7Z7J4, B7Z8Z1, B7Z9C1, C9J521, |
| UniProt Related Accession: | O15350 |
| Molecular Weight: | 61,694 Da |
| NCBI Full Name: | Tumor protein p73 |
| NCBI Synonym Full Names: | tumor protein p73 |
| NCBI Official Symbol: | TP73 |
| NCBI Official Synonym Symbols: | P73 |
| NCBI Protein Information: | tumor protein p73 |
| UniProt Protein Name: | Tumor protein p73 |
| UniProt Synonym Protein Names: | p53-like transcription factor; p53-related protein |
| Protein Family: | Tumor protein |
| UniProt Gene Name: | TP73 |
| 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-p73 (Phospho-Tyr99) Antibody, Anti-p73 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.)
