Description
Tryptophan Hydroxylase (Phospho-Ser260)Colorimetric Cell-Based ELISA Kit
The Tryptophan Hydroxylase Phospho-Ser260 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the accurate detection of phosphorylated Ser260 levels of tryptophan hydroxylase in cell lysates and tissue extracts. This kit offers high sensitivity and specificity, allowing for precise and reliable measurements, making it an ideal tool for various research applications.Tryptophan hydroxylase is a key enzyme involved in the biosynthesis of serotonin, a neurotransmitter that plays a critical role in mood regulation, sleep, and other physiological processes. Phosphorylation of tryptophan hydroxylase at Ser260 has been implicated in the regulation of its activity, making it a key target for studying the serotonin pathway and its role in various diseases, including depression, anxiety, and other mental health disorders.
By providing researchers with a comprehensive and easy-to-use assay for measuring phosphorylated Ser260 levels of tryptophan hydroxylase, this ELISA kit enables a deeper understanding of serotonin biosynthesis and its implications for human health. It is an essential tool for advancing research in the fields of neuroscience, pharmacology, and psychiatry, and holds great promise for the development of novel therapeutics targeting the serotonin pathway.
| Product Name: | Tryptophan Hydroxylase (Phospho-Ser260)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01441 |
| ELISA Type: | Cell-Based |
| Target: | Tryptophan Hydroxylase (Phospho-Ser260) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The Tryptophan Hydroxylase (Phospho-Ser260) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Tryptophan Hydroxylase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Tryptophan Hydroxylase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Tryptophan Hydroxylase phosphorylation.
Qualitative determination of Tryptophan Hydroxylase (Phospho-Ser260) concentration is achieved by an indirect ELISA format. In essence, Tryptophan Hydroxylase (Phospho-Ser260) is captured by Tryptophan Hydroxylase (Phospho-Ser260)-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: 7166, UniProt ID: P17752, OMIM: 191060, Unigene: Hs.591999 |
| Gene Symbol: | TPH1 |
| Sub Type: | Phospho |
| UniProt Protein Function: | TPH1: an enzyme that is rate-limiting in the biosynthesis of serotonin. It catalyzes the biopterin dependent monooxygenation of tryptophan to 5-hydroxytryptophan (5HT), which is subsequently decarboxylated to form the neurotransmitter serotonin. Its expression is limited to a few specialized tissues: raphe neurons, pinealocytes, mast cells, mononuclear leukocytes, beta-cells of the islets of Langerhans, and intestinal and pancreatic enterochromaffin cells. Two alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Amino Acid Metabolism - tryptophan; EC 1.14.16.4; Oxidoreductase Chromosomal Location of Human Ortholog: 11p15.3-p14 Cellular Component: cytosol Biological Process: indolalkylamine biosynthetic process |
| NCBI Summary: | This gene encodes a member of the aromatic amino acid hydroxylase family. The encoded protein catalyzes the first and rate limiting step in the biosynthesis of serotonin, an important hormone and neurotransmitter. Mutations in this gene have been associated with an elevated risk for a variety of diseases and disorders, including schizophrenia, somatic anxiety, anger-related traits, bipolar disorder, suicidal behavior, addictions, and others.[provided by RefSeq, Apr 2009] |
| UniProt Code: | P17752 |
| NCBI GenInfo Identifier: | 116242823 |
| NCBI Gene ID: | 7166 |
| NCBI Accession: | P17752.4 |
| UniProt Secondary Accession: | P17752,O95188, O95189, Q16736, Q3KPG8, D3DQX6, |
| UniProt Related Accession: | P17752 |
| Molecular Weight: | 53,394 Da |
| NCBI Full Name: | Tryptophan 5-hydroxylase 1 |
| NCBI Synonym Full Names: | tryptophan hydroxylase 1 |
| NCBI Official Symbol: | TPH1Â Â |
| NCBI Official Synonym Symbols: | TPRH; TRPHÂ Â |
| NCBI Protein Information: | tryptophan 5-hydroxylase 1 |
| UniProt Protein Name: | Tryptophan 5-hydroxylase 1 |
| UniProt Synonym Protein Names: | Tryptophan 5-monooxygenase 1 |
| Protein Family: | Tryptophan 5-hydroxylase |
| UniProt Gene Name: | TPH1Â Â |
| UniProt Entry Name: | TPH1_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-Tryptophan Hydroxylase (Phospho-Ser260) Antibody, Anti-Tryptophan Hydroxylase 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.)
