TH Antibody, FITC conjugated
Description
Structure and Properties
The TH Antibody, FITC conjugated, is a rabbit-derived polyclonal immunoglobulin G (IgG) antibody conjugated to FITC via primary amine groups (e.g., lysine residues) . Key characteristics include:
Applications in Research
This antibody is widely used for detecting TH in:
Immunofluorescence (IF)
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IHC-P (Paraffin): Localizes TH in fixed tissue sections (e.g., brain, adrenal glands) .
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IHC-F (Frozen) and ICC: Visualizes TH in fresh or cultured cells .
Flow Cytometry
Western Blot (WB)
Dilution Guidelines:
| Method | Dilution Range |
|---|---|
| WB | 1:300–1:5000 |
| IF (IHC-P, IHC-F, ICC) | 1:50–1:200 |
Technical Considerations for Conjugation
FITC conjugation requires precise optimization to balance brightness and specificity:
Optimal Conjugation Conditions
| Parameter | Recommendation |
|---|---|
| pH | 9.5 |
| Temperature | Room temperature (20–25°C) |
| Reaction Time | 30–60 minutes for maximal F/P ratio |
| Protein Concentration | 25 mg/ml (initial) |
Key Challenges:
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Over-conjugation: High FITC-to-antibody ratios (>6) reduce binding affinity and increase background noise .
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Sodium Azide: Must be removed prior to conjugation to prevent FITC inactivation .
Binding Affinity vs. FITC Labeling
Studies show an inverse correlation between FITC-labeling density and antibody affinity :
| FITC Labeling Index | Binding Affinity | Sensitivity | Background Risk |
|---|---|---|---|
| Low (3–4 FITC/Ab) | High | Moderate | Low |
| High (6+ FITC/Ab) | Reduced | High | Increased |
Immunohistochemical Specificity
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Higher FITC labeling improves signal intensity but may compromise specificity, necessitating titration for optimal results .
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DEAE Sephadex chromatography separates optimally labeled antibodies from under- or over-labeled fractions .
Comparative Data: FITC-Conjugated Antibodies
The table below highlights examples of FITC-conjugated antibodies and their applications:
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- These results provide a novel mechanism of how nitric oxide (NO) can modulate TH's enzymatic activity through S-nitrosylation. PMID: 28287127
- It is a genetic risk factor for Parkinson's disease. PMID: 29724574
- One novel mutation of c.679A>G (p.T227A) in GCH1 and 3 known mutations of c.457C>T (p.R153X), c.739G>A (p.G247S), and c.698G>A (p.R227H) in tyrosine hydroxylase (TH) have been found and predicted to be damaging or deleterious. PMID: 29405179
- This study does not support the hypothesis that early-onset Parkinson's disease may be the male presentation of TH deficiency attributed to this founder mutation in Greek patients. PMID: 27666733
- A novel heterozygous variant in tyrosine hydroxylase was identified in Chinese patients with dopa-responsive dystonia. PMID: 27619486
- This study indicates that mutations in TH are uncommon in late-onset Parkinson's disease. PMID: 27185167
- The purpose of this study is to investigate the clinical significance of tyrosine hydroxylase (TH) expression in peripheral blood (PB) at diagnosis in patients with neuroblastoma. The treatment intensity should be tailored according to TH expression in PB at diagnosis. PMID: 27034145
- Our results suggest that the TH-immunoreactive cells in the human cortex do not overlap with any known neurochemically-defined subsets of interneurons and provide further evidence of differences in the phenotype of these cells across species. PMID: 27448941
- Results show that the positive rates and expression levels of nestin, tyrosine hydroxylase (TH), GFAP, and IL-17 were significantly decreased while Foxp3 and the ratio of Foxp3/IL-17 were statistically elevated in bone marrow (BM) of acute myeloid leukemia (AML) patients. PMID: 27016413
- Data suggest that TH phosphorylated at Ser-31 co-distributes with Golgi complexes and synaptic-like vesicles in rat and human dopaminergic neurons/cell lines; Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. PMID: 28637871
- TH is a robust interaction partner of different 14-3-3 dimer types with moderate variability between the 14-3-3 dimers on their regulation of TH. PMID: 26825549
- Germline mutations in the TH gene are linked to Familial isolated pituitary adenoma in a Brazilian Family. PMID: 27245436
- No statistically significant differences were found between cases and controls for the allele frequencies in five genes: TH, SLC18A2, DRD1, DRD3, and COMT. Conversely, some alleles of the 12 SNPs from the DRD2 locus and the 5 from the MAOA locus showed significant associations with excessive alcohol consumption. PMID: 26447226
- Results show that metastasis-associated protein 1 (MTA1) and tyrosine hydroxylase (TH) levels were significantly down-regulated in Parkinson disease (PD) samples as compared with normal brain tissue PMID: 27044752
- The reduction of tyrosine hydroxylase-immunoreactive neurons occurring in the locus coeruleus after perinatal hypoxic insults persists into adulthood PMID: 26647061
- The data suggest that the presence of a homozygous V81M polymorphism is associated with more severe freezing of gait in patients with Parkinson's disease PMID: 26732803
- In this study, we found that TH protein levels did not differ between control and schizophrenia groups in the nucleus accumbens. PMID: 26386900
- In high-risk metastatic Neuroblastoma, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses. PMID: 26498952
- The allelic frequency of the TH01 marker in 171 Swiss sudden infant death syndrome (SIDS) infants and 500 healthy and gender-matched Caucasian adults showed that the 9.3 allele is similarly distributed in SIDS cases and controls (27.2% vs. 25.6%; p-value = 0.562). PMID: 24975687
- This study showed a new tyrosine hydroxylase knock-in mouse model of l-DOPA-responsive dystonia. PMID: 26220941
- The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to a decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. PMID: 26276013
- Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research. PMID: 25462571
- A detailed analysis of the interaction between singly or doubly phosphorylated human tyrosine hydroxylase isoform 1(1-50) peptides and 14-3-3zeta PMID: 25418103
- Study found evidence that DNA variation in the ADRA2A gene may be causally related to ADHD-like behaviors, and for a novel association between a TH gene variant and intra-individual variability PMID: 24166412
- Proteomics analysis show that Ser40 of TH protein does not significantly contribute to the binding of 14-3-3gamma, and rather has reduced accessibility in the TH:14-3-3gamma complex. PMID: 24947669
- Increased expression of TH and GAP43 might be a molecular mechanism for left atrial myoelectricity remodeling of aging atrial fibrillation patients, which might be potential therapeutic targets of atrial fibrillation. PMID: 24301786
- Biosynthesis of catecholamine by the action of TH should be deeply involved in decreased intellectual ability in patients with schizophrenia PMID: 24417771
- A297, E362/E365 and S368 of TH were shown to mediate high affinity dopamine inhibition through V(max) reduction and increasing the K(M) for the cofactor. PMID: 24334288
- Tyrosine hydroxylase polymorphisms contribute to attempted suicide in schizophrenia. PMID: 24275212
- Neurons of the substantia nigra from the Lesch-Nyhan disease cases show reduced melanization and reduced reactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. PMID: 24891139
- Achilles tendon tenocytes produce tyrosine hydroxylase. PMID: 22292987
- In a South African cohort, Africans had a higher incidence of hypertension and higher occurrence of the C-824T TH mutation. However, the contribution of the tyrosine hydroxylase C-824T polymorphism to hypertension could not be confirmed. PMID: 23489065
- Nurr1 overexpression significantly increased the SIRT1 occupancy of the consensus elements for Nurr1 binding hTH promoter region. PMID: 23977047
- The region surrounding pSer19 of Tyrosine hydroxylase adopts an extended conformation in the 14-3-3gamma-bound state, whereas it adopts a bent conformation when free in solution. PMID: 24055376
- Data suggest that coordination of nitric oxide to Fe(II) in TyrH is directed by the presence of tetrahydropterin at the active site, binding in a fashion that may be important for directing the first step of the catalytic cycle toward hydroxylation of tyrosine. PMID: 24168553
- In 10 sporadic cases of dopa-responsive dystonia, only two heterozygous tyrosine hydroxylase mutations (Ser19Cys and Gly397Arg) were found in two subjects with unknown pathogenicity. PMID: 23762320
- Data indicate that the C-terminal domain was the immunodominant part of tryptophan hydroxylase TPH1, the epitopes of tryptophan hydroxylase TPH2 and tyrosine hydroxylase (TH) were mainly located in the N-terminal regulatory domains. PMID: 23182718
- Our studies have clearly identified a glucocorticoid-responsive element in a 7 bp AP-1-like motif in the promoter region at -7.24 kb of the human TH gene PMID: 23647419
- In severe prolonged fetal hypoxia, there was a striking reduction or absence of tyrosine hydroxylase in all the mesencephalic nuclei. PMID: 23481708
- This review discusses the current understandings on the genetic variants in TH and their correlations with Parkinson's disease. PMID: 22583432
- This study presented a THD family with predominant myoclonus-dystonia and a new genotype. PMID: 22815559
- Molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124G>C in the TH gene PMID: 22691284
- mRNA expressions of AQP4 and TH were found to be reduced whereas that of PBP was found to be elevated when compared with those of healthy control samples PMID: 22083667
- Data show calbindin (CB)- and tyrosine hydroxylase (TH)-cells were distributed in the three striatal territories, and the density of calretinin (CR) and parvalbumin (PV) interneurons were more abundant in the associative and sensorimotor striatum. PMID: 22272358
- Protein levels for tyrosine hydroxylase peaked during the first year of life and then gradually declined to adulthood. PMID: 22336227
- Data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene PMID: 21815951
- These results suggest that region-specific methylation and methyl-CpG binding domain proteins play important roles in TH gene regulation in neural stem cells. PMID: 22001923
- Human RXRalpha interacts with and represses Nurr1-dependent transcriptional activation in tyrosine hydroxylase (TH)-expressing dopaminergic neuronal stem cells in culture, downregulating TH promoter activity. PMID: 22066143
- Data indicate that TH gene expression can be regulated by alpha-synuclein (alpha-SYN); further, interference with TH gene expression through elevated levels of alpha-SYN could be associated with dopaminergic neuronal dysfunction. PMID: 21656370
- Data from samples of centenarians, nonagenarians, and younger controls suggest that the TH01 STR locus exhibits no significant influence on the ability of attaining exceptional old age in Germans. PMID: 21407269
Q&A
What is the functional significance of TH in neurobiological research, and how does FITC conjugation enhance its detection?
Tyrosine hydroxylase catalyzes the rate-limiting step in dopamine, norepinephrine, and epinephrine biosynthesis, making it a critical marker for dopaminergic neurons and adrenal chromaffin cells . FITC conjugation enables fluorescence-based detection in techniques like immunofluorescence (IF) and immunohistochemistry (IHC), providing spatial resolution of TH expression in tissue sections or cultured neurons.
Methodological Consideration:
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Antibody Validation: Ensure specificity via knockout tissue controls or siRNA-mediated TH knockdown .
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Conjugation Quality: FITC labeling indices (moles of FITC per antibody molecule) >4 may reduce antigen binding affinity due to steric hindrance . Validate using dot-blot assays comparing labeled vs. unlabeled antibody reactivity .
How should researchers optimize TH-FITC antibody dilution for western blotting versus immunofluorescence?
Optimal dilution depends on antibody affinity, target abundance, and detection system sensitivity.
Validation Step: Perform checkerboard titrations using positive controls (e.g., substantia nigra lysates for WB, midbrain sections for IF) .
What controls are essential when using TH-FITC antibodies to avoid false positives in immunohistochemistry?
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Primary Controls:
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Tissue-Specific Controls:
How does FITC labeling index influence TH antibody performance in multiplexed imaging assays?
FITC labeling index (LI) critically impacts assay specificity and sensitivity:
| Labeling Index (LI) | Binding Affinity (KD) | Non-Specific Staining Risk | Ideal Use Case |
|---|---|---|---|
| 1–3 | <10 nM | Low | Quantitative confocal imaging |
| 4–6 | 10–50 nM | Moderate | High-throughput screening |
| >6 | >50 nM | High | Avoid for precise localization |
Data derived from FITC-antibody characterization studies .
Mitigation Strategy: For multiplex assays pairing TH-FITC with other fluorophores, use LI ≤3 and spectral unmixing algorithms to minimize crosstalk .
What experimental approaches resolve contradictions between TH-FITC staining patterns and qPCR data?
Discrepancies often arise from post-transcriptional regulation or antibody cross-reactivity.
Stepwise Analysis:
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Technical Validation:
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Biological Validation:
How can researchers quantify TH-FITC signal intensity in dynamic processes like neuroregeneration?
Ratiometric Analysis Protocol:
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Internal Standard: Co-stain with a spectrally distinct antibody (e.g., Cy5-conjugated NeuN) for normalization .
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Image Acquisition:
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Use consistent exposure times across samples.
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Capture z-stacks to account for tissue thickness variability.
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Data Expression:
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Calculate FITC:Cy5 ratio to control for section-to-section variability.
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Pitfall Avoidance: Ensure linear detection range by plotting signal intensity vs. exposure time .
What strategies improve TH-FITC antibody performance in fixed frozen versus formalin-fixed tissues?
| Tissue Type | Fixation Artifact Mitigation | Antigen Retrieval Method |
|---|---|---|
| Frozen Sections | Acetone fixation ≤10 min | 0.1% Triton X-100 permeabilization |
| Formalin-Fixed | 24 hr post-fixation in 4% PFA | Citrate buffer (pH 6.0, 95°C, 30 min) |
Validation: Compare TH-FITC signal with tyramide signal amplification (TSA) to confirm epitope accessibility .
How should non-specific binding be addressed when using TH-FITC in autofluorescence-rich tissues (e.g., adrenal medulla)?
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Chemical Quenching: Treat sections with 0.3% Sudan Black B (10 min) to suppress lipofuscin autofluorescence .
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Cross-Absorption: Preabsorb antibody with adrenal lysate (1 mg/mL, 2 hr) to remove non-specific IgGs .
What are the implications of TH-FITC batch variability for longitudinal studies?
Critical Parameters:
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Lot-to-Lot Consistency: Assess via ELISA against recombinant TH (EC50 shift >2-fold indicates incompatibility) .
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Normalization: Include a reference sample (e.g., commercial TH-positive control slides) in each experiment .
Can TH-FITC antibodies be used for live-cell imaging of dopaminergic neurons?
Feasibility and Limitations:
