Description
TEBP (Phospho-Ser113)Colorimetric Cell-Based ELISA Kit
The TEBP Phospho-Ser113 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the accurate detection and quantification of TEBP (Telomere End-Binding Protein) phosphorylated at serine 113 in cell lysates. This innovative kit offers high sensitivity and specificity, providing researchers with reliable and reproducible results for a variety of experimental settings.TEBP is a critical protein involved in telomere maintenance and DNA repair mechanisms, making it a key player in cellular health and longevity. The phosphorylation of TEBP at serine 113 has been implicated in regulating its function, highlighting the importance of studying this modification in cellular processes and disease pathways.
By utilizing the TEBP Phospho-Ser113 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of TEBP phosphorylation in various biological processes and disease conditions. This kit is an essential tool for scientists working in the fields of molecular biology, cell signaling, and cancer research, offering a new level of precision and accuracy in their experimental workflows.
| Product Name: | TEBP (Phospho-Ser113)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01324 |
| ELISA Type: | Cell-Based |
| Target: | TEBP (Phospho-Ser113) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The TEBP (Phospho-Ser113) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TEBP protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated TEBP in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on TEBP phosphorylation.
Qualitative determination of TEBP (Phospho-Ser113) concentration is achieved by an indirect ELISA format. In essence, TEBP (Phospho-Ser113) is captured by TEBP (Phospho-Ser113)-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: 10728, UniProt ID: Q15185, OMIM: 607061, Unigene: Hs.50425 |
| Gene Symbol: | PTGES3 |
| Sub Type: | Phospho |
| UniProt Protein Function: | TEBP: Molecular chaperone that localizes to genomic response elements in a hormone-dependent manner and disrupts receptor- mediated transcriptional activation, by promoting disassembly of transcriptional regulatory complexes. Belongs to the p23/wos2 family. |
| UniProt Protein Details: | Protein type:Transferase; Chaperone; Nuclear receptor co-regulator; EC 5.3.99.3; Isomerase Chromosomal Location of Human Ortholog: 12q13.3|12 Cellular Component: chromosome, telomeric region; cytoplasm; cytosol; nucleoplasm; nucleus; telomerase holoenzyme complex Molecular Function:prostaglandin-E synthase activity; protein binding; telomerase activity; unfolded protein binding Biological Process: arachidonic acid metabolic process; cyclooxygenase pathway; prostaglandin biosynthetic process; RNA-dependent DNA replication; signal transduction; telomere maintenance |
| NCBI Summary: | This gene encodes an enzyme that converts prostaglandin endoperoxide H2 (PGH2) to prostaglandin E2 (PGE2). This protein functions as a co-chaperone with heat shock protein 90 (HSP90), localizing to response elements in DNA and disrupting transcriptional activation complexes. Alternative splicing results in multiple transcript variants. There are multiple pseudogenes of this gene on several different chromosomes. [provided by RefSeq, Feb 2016] |
| UniProt Code: | Q15185 |
| NCBI GenInfo Identifier: | 8928247 |
| NCBI Gene ID: | 10728 |
| NCBI Accession: | Q15185.1 |
| UniProt Secondary Accession: | Q15185,Q8WU70, A8K7D0, B4DHP2, B4DP11, B4DP21, |
| UniProt Related Accession: | Q15185 |
| Molecular Weight: | 16,476 Da |
| NCBI Full Name: | Prostaglandin E synthase 3 |
| NCBI Synonym Full Names: | prostaglandin E synthase 3 |
| NCBI Official Symbol: | PTGES3 |
| NCBI Official Synonym Symbols: | P23; TEBP; cPGES |
| NCBI Protein Information: | prostaglandin E synthase 3 |
| UniProt Protein Name: | Prostaglandin E synthase 3 |
| UniProt Synonym Protein Names: | Cytosolic prostaglandin E2 synthase; cPGES; Hsp90 co-chaperone; Progesterone receptor complex p23; Telomerase-binding protein p23 |
| UniProt Gene Name: | PTGES3 |
| UniProt Entry Name: | TEBP_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-TEBP (Phospho-Ser113) Antibody, Anti-TEBP 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.)
