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Ordering Information
- : MAB5278
- : 500 µg
Anti-Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase, clone PH8
| Species Reactivity | Key Applications | Host | Format | Antibody Type |
|---|---|---|---|---|
| H | IH, IP, WB | Mouse | Purified | Monoclonal Antibody |
Description:
Anti-Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase, clone PH8
Trade Name:
Chemicon (Millipore)
Clone:
PH8
Isotype:
IgG1
Background Information:
PH8 Antibody is a murine monoclonal antibody that binds a common epitope of Tryptophan hydroxylase (TRH), Tyrosine hydroxylase (TYH) and Phenylalanine hydroxylase (PAH).1 In the literature this monoclonal antibody is cited as PH8. Tyrosine hydroxylase (TYH) is the enzyme which converts tyrosine to dihydroxyphenylalanine (L-dopa), a precursor of the catecholamine neurotransmitters dopamine, noradrenaline and adrenaline. Tryptophan hydroxylase (TRH) is the enzyme that converts 5-hydroxytryptophan to serotonin. In fresh tissue the PH8 Antibody binds to TYH and TRH so is a marker for TYH-containing catecholaminergic neurons,2 and serotonergic neurons.3,4,5 Tryptophan hydroxylase (TRH) can be used as a marker for serotonin as it converts 5-hydroxy-tryptophan to serotonin. Serotonin is rapidly metabolised and is unable to be detected by anti-serotonin antibodies in post mortem tissue. In human tissue that has been formalin-fixed, due to a change in the antigenic determinant of tyrosine hydroxylase (TYH), PH8 Antibody will bind only tryptophan hydroxylase (TRH).3,4,5 The PH8 Antibody can therefore be used to specifically identify serotonergic neurons in fixed human tissue. Phenylalanine hydroxylase (PAH) can be detected in hepatic tissue sections using the PH8 Antibody.
Species Reactivity:
Human
Application Notes:
The PH8 Antibody can be used to identify dopaminergic and serotonergic neurons by immuno-histochemistry and for Western blot analysis, immunoprecipitation and immuno-histochemistry of TYH, PAH and TRH.
PROTOCOLS
Immunohistochemistry
The PH8 Antibody can be used for the immunohistochemical detection of dopaminergic and serotonergic neurons in human and rat brain stem tissue.
1. Tissue should be formalin fixed and stored in formalin prior to use for a minimum of five days.
2. Cryoprotect tissue using 30% sucrose in 0.1M Tris pH 7.4 buffer for 24-72 hours.
3. Cut using a sledge microtome to 50 μm thickness.
4. Wash the tissue samples using Tris buffer prior to commencing the staining procedure.
5. Treat the tissue samples for 3 x 15 minutes in 50% alcohol.
6. Treat the tissue samples for 20 minutes in 50% alcohol and 3% H2O2.
7. Treat tissue for 20 minutes with 10% normal horse serum in Tris buffer. This acts to block endogeneous H2O2 staining.
8. Dilute the PH8 Antibody in Tris buffer, add to the tissue samples and incubate for 1-3 days. Recommended dilutions are 1:2,000-1:10,000. At high antibody concentration all monoaminergic neurons are stained with no distinction between serotonegic and catecholominergic cells. By diluting the antibody concentrate, cell types are distinguishable due to variation in staining intensity. At lower concentrations (1:5,000-1:10,000 dilution) only serotonergic cells will stain.
9. Wash tissue (3 x 15 minutes), then add a biotinylated anti-mouse secondary antibody and incubate on an orbital shaker at room temperature (RT) for 1 hour.
10. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 1 hour with the tertiary complex (ELITE KIT, Vector, USA).
11. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 10 minutes with Tris buffered diamino-benzidine substrate. Add 0.1% H2O2 and incubate on an orbital shaker at RT for a further 5 minutes.
12. Mount tissue onto gelatinised slides and allow to dry prior to microscopy.
PROTOCOLS
Immunohistochemistry
The PH8 Antibody can be used for the immunohistochemical detection of dopaminergic and serotonergic neurons in human and rat brain stem tissue.
1. Tissue should be formalin fixed and stored in formalin prior to use for a minimum of five days.
2. Cryoprotect tissue using 30% sucrose in 0.1M Tris pH 7.4 buffer for 24-72 hours.
3. Cut using a sledge microtome to 50 μm thickness.
4. Wash the tissue samples using Tris buffer prior to commencing the staining procedure.
5. Treat the tissue samples for 3 x 15 minutes in 50% alcohol.
6. Treat the tissue samples for 20 minutes in 50% alcohol and 3% H2O2.
7. Treat tissue for 20 minutes with 10% normal horse serum in Tris buffer. This acts to block endogeneous H2O2 staining.
8. Dilute the PH8 Antibody in Tris buffer, add to the tissue samples and incubate for 1-3 days. Recommended dilutions are 1:2,000-1:10,000. At high antibody concentration all monoaminergic neurons are stained with no distinction between serotonegic and catecholominergic cells. By diluting the antibody concentrate, cell types are distinguishable due to variation in staining intensity. At lower concentrations (1:5,000-1:10,000 dilution) only serotonergic cells will stain.
9. Wash tissue (3 x 15 minutes), then add a biotinylated anti-mouse secondary antibody and incubate on an orbital shaker at room temperature (RT) for 1 hour.
10. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 1 hour with the tertiary complex (ELITE KIT, Vector, USA).
11. Wash tissue (3 x 15 minutes) and incubate on an orbital shaker at RT for 10 minutes with Tris buffered diamino-benzidine substrate. Add 0.1% H2O2 and incubate on an orbital shaker at RT for a further 5 minutes.
12. Mount tissue onto gelatinised slides and allow to dry prior to microscopy.
Presentation:
The immunoglobulin fraction has been purified from ascites fluid by Protein G chromatography. 500μg liquid protein at 2mg/mL in phosphate buffered saline (PBS). The product is supplied sterile-filtered through a 0.22 micron filter.
Storage Conditions:
The PH8 Antibody is shipped liquid at ambient temperature. Liquid material is stable for at up to 6 months when stored at 2-8°C.
UniProt Number:
Entrez Gene Number:
Gene Symbol:
- TPH1
- TPRH
- MGC119994
- TPH
- TRPH
- EC 1.14.16.4
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
This gene encodes a member of the pterin-dependent aromatic acid hydroxylase family. The encoded protein catalyzes the first and rate limiting step in the biosynthesis of serotonin, an important hormone and neurotransmitter. The human genome contains two related tryptophan hydroxylases, one on chromosome 11p15-p14 and one on chromosome 12q21. This gene is expressed predominantly in the gut, pineal gland, spleen, and thymus.
Key Applications:
- Immunohistochemistry
- Immunoprecipitation
- Western Blotting
UniProt Summary:
FUNCTION: SwissProt: P07101 # Plays an important role in the physiology of adrenergic neurons.
COFACTOR: Fe(2+) ion.
SIZE: 528 amino acids; 58524 Da
TISSUE SPECIFICITY: Mainly expressed in the brain and adrenal glands.
DISEASE: SwissProt: P07101 # Defects in TH are the cause of autosomal recessive Segawa syndrome [MIM:605407]; also known as DOPA-responsive dystonia. Typically, it begins in childhood or adolescence with progressive difficulty in walking and, in some cases, spasticity. Some cases present with parkinsonian symptoms in infancy and are referred to as autosomal recessive infantile parkinsonism.
SIMILARITY: SwissProt: P07101 ## Belongs to the biopterin-dependent aromatic amino acid hydroxylase family.
COFACTOR: Fe(2+) ion.
SIZE: 528 amino acids; 58524 Da
TISSUE SPECIFICITY: Mainly expressed in the brain and adrenal glands.
DISEASE: SwissProt: P07101 # Defects in TH are the cause of autosomal recessive Segawa syndrome [MIM:605407]; also known as DOPA-responsive dystonia. Typically, it begins in childhood or adolescence with progressive difficulty in walking and, in some cases, spasticity. Some cases present with parkinsonian symptoms in infancy and are referred to as autosomal recessive infantile parkinsonism.
SIMILARITY: SwissProt: P07101 ## Belongs to the biopterin-dependent aromatic amino acid hydroxylase family.
Qty/Pk:
500 µg
Brand Family:
Chemicon
Format:
Purified
Host:
Mouse
Product Name:
Anti-Tryptophan Hydroxylase/Tyrosine Hydroxylase/Phenylalanine Hydroxylase, clone PH8
Antibody Type:
Monoclonal Antibody