ARX Antibody, FITC conjugated
Description
Biochemical Properties and Mechanism
ARX Protein:
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Function: Regulates neuronal migration, interneuron differentiation, and pancreatic alpha-cell development .
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Structure: Contains a homeobox DNA-binding domain, enabling interaction with regulatory sequences like 5’-TAATTA-3’ .
FITC Conjugation:
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Excitation/Emission: 495 nm (excitation), 519 nm (emission) .
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Conjugation Chemistry: FITC binds lysine residues via isothiocyanate groups, retaining antibody specificity .
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Impact on Antibody:
| FITC Properties | Details |
|---|---|
| Quantum Yield | High (~0.93) |
| Photostability | Moderate (prone to photobleaching) |
| Multiplex Compatibility | Compatible with TRITC, Cy3, Texas Red, Cy5 |
Applications in Research
ARX FITC-conjugated antibodies are used in:
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Immunofluorescence (IF): Visualize ARX in fixed cells or tissues (e.g., pancreatic islets) .
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Flow Cytometry: Quantify ARX expression in single-cell suspensions .
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Western Blot (WB): Detect ARX at ~58 kDa in lysates (e.g., mouse brain) .
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Multiplex Imaging: Combine with Opal™ dyes (e.g., Opal 520/570) for co-localization studies .
Key Studies:
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Neuronal Migration: FITC-labeled ARX antibodies identified mislocalized interneurons in Arx mutation models .
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Pancreatic Development: Demonstrated ARX’s role in alpha-cell specification using IHC .
Validation Data and Research Findings
Specificity:
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Western Blot: Single band at 58 kDa in human, mouse, and rat samples .
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Immunohistochemistry: Nuclear staining in human brain and pancreatic tissues .
| Validation Assay | Results | Source |
|---|---|---|
| WB (Human Jurkat) | Clear 58 kDa band | |
| IF (Rat Neurons) | Nuclear localization in GABAergic interneurons | |
| IHC (Human Pancreas) | Specific alpha-cell staining |
Functional Insights:
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Gene Regulation: ARX activates KDM5C transcription, influencing histone demethylation .
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Disease Links: ARX mutations cause X-linked intellectual disability and epilepsy .
Considerations for Experimental Use
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Labeling Index: Use antibodies with moderate FITC labeling to avoid affinity loss .
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Controls: Include secondary-only and isotype controls to exclude non-specific binding .
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Storage: Protect from light; store at -20°C in 50% glycerol .
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Photobleaching: Limit exposure time or use antifade mounting media .
Product Specs
Target Background
- ARX gene mutation c.88G>T is associated with X-linked intellectual disability. PMID: 29896742
- ARX is not associated with endometriosis and is not a suitable biomarker for ovarian endometriosis. While present in ovarian stroma and derived cells, as well as in sex cord-stromal tumors of the ovary, it lacks diagnostic utility in this context. PMID: 29275192
- Arx and Dnmt1 are sufficient for the targeted generation of beta cells from adult pancreatic alpha cells. PMID: 28215845
- This review emphasizes the importance of ARX screening in both male and female patients presenting with intellectual disability, seizures, and particularly, complete agenesis of the corpus callosum (ACC). PMID: 28150386
- A core pathway involving transcription regulators, including Hdac4 (involved in chromatin condensation and transcriptional repression), and its target, the transcription factor Twist1, are suggested as potential drivers of intellectual disability and infantile spasms in patients with ARX polyalanine expansion mutations. PMID: 27798109
- ARX inhibition in expanded alpha-cell-derived cells treated with retinoic acid did not induce transdifferentiation into insulin-producing cells. PMID: 26856418
- A novel c.34G>T (p.(E12*)) variant in the ARX gene was identified in two male patients with early-onset infantile spasms. This variant resulted in reinitiation of mRNA translation at the next AUG codon. PMID: 26306640
- ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon, and insulin-positive cells derived from human embryonic stem cells. PMID: 26633894
- This study suggests that some structural and behavioral anomalies observed in patients with ARX mutations are specifically due to alterations in pallial progenitor function. PMID: 24794919
- Neurodevelopmental disturbances in patients may not solely result from increased ARX dosage due to duplication. PMID: 26337422
- An expansion of seven alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation, indicating that ARX/Arx is necessary for the specification of a subset of enteroendocrine cells in both humans and mice. PMID: 25171319
- ARX duplication does not invariably lead to detrimental effects on brain development, unlike ARX haploinsufficiency. PMID: 25044608
- The ARX c.429_452dup24 mutation may serve as a developmental model for limb kinetic apraxia. PMID: 24528893
- A non-malformation phenotype with intellectual disability and dystonia is caused by an ARX missense mutation outside the polyalanine tract-coding regions. PMID: 23657928
- This study demonstrated that ARX polyalanine expansion modifies glutamatergic neuron excitability and morphology without affecting GABAergic neuron development. PMID: 22628459
- Combined aberrations of CDKL5 and ARX are an important consideration in the genetic forms of early-onset epilepsy in boys. PMID: 23583054
- This research reveals a novel and conserved role of Arx in mammalian endocrine cell development. PMID: 22387004
- A significant deviation from the expected Mendelian 1:1 ratio of transmission was observed, favoring the c.429_452dup ARX mutation. PMID: 22490986
- Mutations in the Aristaless-Related Homeobox gene (ARX) have been linked to X-linked Infantile Spasms Syndrome. PMID: 22565167
- A novel ARX mutation in three brothers was associated with mild developmental delay and early hand preference. PMID: 22922607
- A patient in a Turkish family with non-syndromic X-linked mental retardation exhibited an abnormal band pattern on agarose gel electrophoresis; sequence analysis revealed the c.428_451dup(24bp) mutation in exon 2 of ARX. PMID: 23072184
- Missense mutations in the ARX homeodomain represent loss-of-function mutations, leading to reduced or complete loss of DNA binding and consequently, transcriptional repression. PMID: 22194193
- Molecular analysis of ARX mutations should be considered a routine diagnostic procedure for males with either syndromic or non-syndromic X-linked intellectual disability (XLID). PMID: 22642246
- ARX homeodomain mutations result in loss of both DNA binding and transcriptional repression activity. PMID: 22252899
- Homeodomain mutations' protein mislocalization correlated with clinical severity of non-syndromic intellectual disability. PMID: 21496008
- This study confirmed the critical role of the aristaless-related homeobox in the pathogenesis of epileptic encephalopathies. PMID: 21482751
- This study describes a novel ARX mutation in a family, causing Ohtahara syndrome with abnormal genital and psychomotor development (OAGPD) in a male infant and neurocognitive/psychiatric manifestations in heterozygous female carriers. PMID: 21426321
- ARX polyalanine expansions are primarily associated with syndromic mental retardation. PMID: 21204215
- Contractions in the second polyalanine tract of ARX are rare, non-pathogenic polymorphisms. PMID: 21204226
- Two male individuals, born to monozygotic twin sisters, presented with Ohtahara syndrome progressing to West syndrome and epileptic encephalopathy; a previously unreported missense mutation (c.1604T>A) in exon 5 of the ARX gene was identified in both children. PMID: 21108397
- ARX contributes to both endocrine and exocrine development of the human pancreas; its deficiency can lead to severe phenotypes observed in X-linked lissencephaly with abnormal genitalia patients. PMID: 20538404
- Novel frameshift mutations (Ala524fsX534 and E536fsX672) in the terminal exon of the ARX gene were identified in two Ohtahara syndrome patients (aged 2 and 13 years) from two families. PMID: 20384723
- This review provides a catalog of known ARX mutations and associated clinical phenotypes. PMID: 20506206
- Three cases of mental retardation in two families were found to have the c.428_451dup24 mutation in the ARX gene during X-fragile syndrome screening. PMID: 19085879
- These findings expand the spectrum of clinical phenotypes associated with ARX gene mutations and highlight the molecular pathogenic effects of individual mutations. PMID: 19738637
- ARX expression is specific to the telencephalon and thalamus. Mutations cause mental retardation without brain malformations. PMID: 11971879
- Data suggest that ARX gene mutations are significant causes of mental retardation, often accompanied by diverse neurological manifestations. PMID: 12142061
- A novel syndrome of X-linked myoclonic epilepsy with generalized spasticity and intellectual disability in boys (XMESID) was described, with a novel missense mutation (1058C>T) identified in the ARX open reading frame. PMID: 12177367
- The expression pattern indicates that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the central nervous system. PMID: 12359145
- ARX plays a role in X-linked lissencephaly with abnormal genitalia. PMID: 12379852
- Disruption of the STK9 gene causes severe X-linked infantile spasms and mental retardation. PMID: 12736870
- Two point mutations (790delC & R332C) in two X-linked lissencephaly with abnormal genitalia pedigrees affect the homeodomain of the protein, confirming ARX as a causative gene for XLAG. PMID: 12874405
- A hemizygous 24-bp duplication in exon 2 (441_464dup) results in an expansion from 12 to 20 alanine residues (A155_W156insAAAAAAAA) in the second of four polyalanine tracts in the ARX protein, causing West syndrome. PMID: 12874418
- Thirteen novel mutations were found in the ARX gene in 20 males with X-linked lissencephaly with abnormal genitalia. PMID: 14722918
- Expansions in one of the ARX polyalanine tracts result in nuclear protein aggregation and increased cell death, likely underlying the pathogenesis of associated infantile spasms and mental retardation. PMID: 15533998
- ARX gene mutations can result in various phenotypes, ranging from severe brain malformations to less severe syndromic or non-syndromic forms of X-linked mental retardation. PMID: 15707237
- Familial West syndrome and dystonia are caused by an Aristaless-related homeobox gene (ARX) mutation. PMID: 15726411
- Four nonsyndromic XLMR families had a 24 base pair duplication mutation in exon 2 of ARX. PMID: 15850492
- These results highlight the significant contribution of ARX mutations to X-linked mental retardation (XLMR), suggesting that screening for the c.428-451dup (24 bp) mutation should be considered in all patients with suspected XLMR. PMID: 16523516
- These findings reinforce the importance of ARX mutations in mental retardation, suggesting molecular screening of exon 2 in males with mental retardation of unknown etiology. PMID: 16845484
Q&A
What is the difference between ARX antibody and Arc antibody?
ARX (Aristaless-related homeobox) is a transcription factor important for normal brain development that binds to specific sequence motifs (5'-TAATTA-3') in regulatory elements of target genes . It positively modulates transcription of genes like KDM5C and plays critical roles in neuronal proliferation, interneuronal migration, and differentiation in the embryonic forebrain .
In contrast, Arc (Activity-Regulated Cytoskeleton-Associated Protein) is involved in synaptic plasticity and memory formation. The Arc antibody discussed in the data (ABIN739467) is a polyclonal antibody that targets the amino acid region 101-200 of the Arc protein .
What does FITC conjugation mean for antibody applications?
FITC (Fluorescein Isothiocyanate) conjugation involves crosslinking a primary antibody with the FITC fluorophore using established protocols . This conjugation enables direct visualization of the antibody binding without requiring secondary antibodies, making it valuable for immunofluorescence detection of proteins. FITC-conjugated antibodies can be used in applications such as Western Blotting (WB), Flow Cytometry (FACS), and immunofluorescence microscopy .
What are the key specifications of the FITC-conjugated Arc antibody?
The FITC-conjugated Arc antibody (ABIN739467) is a rabbit polyclonal antibody with binding specificity to amino acids 101-200 of the Arc protein. It shows reactivity with human, rat, and mouse samples, with predicted reactivity for cow, horse, and rabbit. It's purified by Protein A and generated using a KLH-conjugated synthetic peptide derived from human Arc as the immunogen. It has an IgG isotype and is primarily used for Western Blotting and Flow Cytometry applications .
How should researchers optimize protocols for FITC-conjugated antibody applications?
For immunofluorescence applications using FITC-conjugated antibodies:
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Protection from light: FITC-conjugated antibodies should not be exposed to continuous light, which causes gradual loss of fluorescence .
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Optimal dilution: A recommended starting dilution for immunofluorescence on mammalian cells is 1:500 in Phosphate-Buffered Saline (PBS) containing 10% fetal bovine serum (FBS) .
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Blocking procedure: Apply 2 mL of blocking solution (PBS containing 10% FBS) and incubate for 20 minutes at room temperature to reduce non-specific binding .
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Antibody incubation: Remove blocking solution and add 1 mL of PBS/10% FBS containing the FITC-conjugated antibody (1:500 dilution). Incubate for 1 hour at room temperature in the dark .
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Washing and visualization: Wash cells 2 × 5 minutes with PBS and observe with a fluorescence microscope equipped with a FITC filter or appropriate filter setup .
What considerations are important for cross-reactivity assessment?
When using FITC-conjugated antibodies like the Arc antibody (ABIN739467), researchers should:
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Review the documented cross-reactivity: The Arc antibody shows reactivity with human, mouse, and rat samples, with predicted reactivity for cow, horse, and rabbit .
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Perform appropriate controls: Include negative controls (samples lacking the target protein) and positive controls to validate specificity.
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Consider potential off-target binding: Evaluate sequence homology between the immunogen region (AA 101-200 for the Arc antibody) and other proteins in your experimental system.
How does antibody clonality affect experimental design?
The Arc antibody (ABIN739467) is polyclonal , meaning it contains a heterogeneous mixture of antibodies that recognize different epitopes within the target region (AA 101-200). This provides:
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Higher sensitivity: Multiple antibodies binding to different epitopes amplify signal detection
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Increased tolerance to protein denaturation: Recognition of different epitopes means some antibodies may still bind if certain epitopes are modified
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Batch-to-batch variation: May require additional validation between lots
In contrast, monoclonal antibodies (like the ARX antibody EPR27481-32) offer:
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Higher specificity: Recognition of a single epitope
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Consistent reproducibility: Less batch-to-batch variation
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Potentially lower sensitivity: Single epitope binding may produce weaker signals
What are the critical controls for immunofluorescence experiments with FITC-conjugated antibodies?
When conducting immunofluorescence with FITC-conjugated antibodies, implement these controls:
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Negative control: Cells/tissues not expressing the target protein
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Isotype control: A non-specific antibody of the same isotype (IgG for the Arc antibody) and conjugated to FITC
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Unstained sample: To establish baseline autofluorescence
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Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining
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Secondary-only control: For comparative experiments including indirect immunofluorescence
How can researchers conduct effective multiplexing with FITC-conjugated antibodies?
FITC emits green fluorescence (peak emission ~520 nm), allowing for multiplexing with fluorophores of different emission spectra. When designing multiplexed experiments:
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Select complementary fluorophores: Choose fluorophores with minimal spectral overlap with FITC (e.g., Cy5 for Arc antibodies)
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Sequential staining: For multiple primary antibodies from the same host species
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Control for cross-reactivity: Validate each antibody individually before multiplexing
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Appropriate compensation: Adjust for spectral overlap during image acquisition
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Sequential imaging: Consider sequential rather than simultaneous imaging if bleed-through is problematic
What troubleshooting approaches are effective for weak FITC signals?
When experiencing weak fluorescence signals with FITC-conjugated antibodies:
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Prevent photobleaching: Minimize exposure to light during all steps of the protocol
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Optimize antibody concentration: Test different dilutions beyond the recommended 1:500
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Extend incubation time: Consider longer incubation periods (overnight at 4°C)
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Enhance signal detection: Use anti-FITC antibodies conjugated to brighter fluorophores
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Improve sample preparation: Optimize fixation protocols to preserve epitope accessibility
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Test different blocking solutions: The recommended PBS/10% FBS might be suboptimal for certain applications
How should quantitative analysis of FITC-labeled proteins be performed?
For accurate quantification of FITC-labeled proteins:
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Image standardization: Maintain consistent exposure settings between samples
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Background subtraction: Subtract autofluorescence using unstained or isotype controls
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Region of interest (ROI) selection: Define consistent ROIs across samples
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Threshold determination: Establish signal thresholds based on control samples
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Intensity measurement: Measure mean/integrated fluorescence intensity within ROIs
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Normalization: Normalize to cell number, area, or housekeeping protein
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Statistical analysis: Apply appropriate statistical tests based on experimental design
What approaches can resolve discrepancies between antibody-based detection and other methods?
When faced with discrepancies between FITC-conjugated antibody results and other methods:
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Validate antibody specificity: Confirm target recognition using knockout/knockdown controls
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Check antibody epitope: The Arc antibody targets amino acids 101-200 , which may be modified or masked in certain contexts
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Consider protein vs. mRNA discrepancies: For ARX, transcription factor activity may not correlate with transcript levels
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Evaluate post-translational modifications: These may affect antibody binding without altering gene expression
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Test alternative fixation methods: Different fixatives can affect epitope accessibility
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Compare subcellular localization: ARX functions primarily in the nucleus as a transcription factor , while Arc has different subcellular distribution
What are the appropriate applications for FITC-conjugated Arc antibodies versus ARX antibodies?
FITC-conjugated Arc antibody applications:
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Flow cytometry analysis of neuronal activation
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Western blotting for Arc protein expression in synaptic plasticity studies
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Immunofluorescence visualization of activity-dependent Arc expression
ARX antibody applications:
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Immunohistochemistry for brain development studies
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Western blotting for transcription factor expression
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Investigation of neurodevelopmental disorders
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Multiplex immunohistochemistry for co-expression studies
