THOC6 Antibody, HRP conjugated

What is THOC6 and why is it significant in research?

THOC6 is a subunit of the multi-protein THO complex involved in coordination between transcription and mRNA processing. It functions as a critical component of the Transcription Export (TREX) complex, which facilitates mammalian mRNA processing and nuclear export . THOC6 is particularly significant because:

• It facilitates the formation of the TREX tetramer, composed of four THO monomers

• Biallelic pathogenic variants in THOC6 cause THOC6 Intellectual Disability Syndrome (TIDS), also known as Beaulieu-Boycott-Innes Syndrome

• It has recently been identified as a potential biomarker for glioma

The encoded protein contains WD40 repeat domains and is alternatively referred to as WDR58 or functional spliceosome-associated protein 35 (fSAP35) .

What applications is the THOC6 antibody, HRP conjugated suitable for?

The THOC6 antibody, HRP conjugated has been validated for the following applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): Primary application as indicated by the manufacturer

• Western Blotting: Compatible with various cell lines and tissue lysates when used at appropriate dilutions

• Immunohistochemistry: Validated for detection in human tissues with specific antigen retrieval requirements

• Immunoprecipitation: Effective for pulldown assays in certain cell types

For optimal results in each application, researchers should follow the recommended dilution ranges and experimental conditions specified by the manufacturer.

How should THOC6 antibody, HRP conjugated be stored and handled?

For optimal antibody performance and stability:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can damage antibody integrity

• The product is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• For long-term storage beyond routine use, aliquoting is recommended to minimize freeze-thaw cycles

• Working dilutions should be prepared fresh before experiments

What are the recommended dilutions for THOC6 antibody applications?

Different applications require specific antibody dilutions for optimal results:

These ranges provide starting points for optimization. Sample-dependent factors and specific experimental conditions may necessitate further adjustment to achieve optimal signal-to-noise ratios.

How can I validate the specificity of THOC6 antibody in my experimental system?

To confirm antibody specificity:

• Positive controls: Use cell lines or tissues known to express THOC6, such as A431 cells, Neuro-2a cells, or mouse ovary tissue, which have demonstrated positive signals in Western blot applications

• Knockout/knockdown validation: Utilize CRISPR-Cas9 knockout or siRNA-mediated knockdown of THOC6 to confirm specificity. The observed 35 kDa band should be substantially reduced or eliminated in these samples

• Immunogen competition assay: Pre-incubate the antibody with the immunogen peptide before application to samples. This should result in reduced or eliminated signal if the antibody is specific

• Multiple antibody approach: Compare results using different antibodies targeting distinct epitopes of THOC6 to confirm consistent detection patterns

• Cross-species reactivity test: Since the antibody has demonstrated reactivity with human, mouse, and rat samples, comparing detection across species can provide additional validation information

How can THOC6 antibodies be used to investigate THOC6 Intellectual Disability Syndrome (TIDS)?

TIDS is caused by biallelic pathogenic variants in THOC6. To investigate TIDS mechanisms:

• Expression analysis in patient-derived cells:

  • Compare THOC6 protein levels between control, heterozygous carrier, and affected patient-derived iPSCs or neural progenitor cells (NPCs)

  • Western blot analysis can reveal differences in protein abundance, with most TIDS-associated variants showing reduced protein levels

• Subcellular localization studies:

  • Immunofluorescence microscopy using THOC6 antibodies can determine if pathogenic variants alter its nuclear localization

  • Co-staining with markers of nuclear speckles or other nuclear compartments can reveal disrupted localization patterns

• Protein-protein interaction analysis:

  • Immunoprecipitation with THOC6 antibodies followed by Western blotting for TREX complex components (e.g., THOC5, ALYREF) can reveal disrupted interactions

  • Research has shown that biallelic THOC6 variants reduce the binding affinity of ALYREF to THOC5 without affecting protein expression of TREX members

• Corticogenesis model systems:

  • THOC6 antibodies can be used to analyze protein expression in developing neural tissues

  • Studies have demonstrated that THOC6 is required for key signaling pathways regulating the transition from proliferative to neurogenic divisions during human corticogenesis

What methodologies can reveal THOC6's role in neural mRNA processing?

THOC6 has been implicated in mRNA processing crucial for neural development:

• mRNA export analysis:

  • Compare nuclear/cytoplasmic ratios of polyA+ mRNA in control versus THOC6-deficient neural cells

  • While global mRNA export may not show significant alterations in THOC6-affected neural progenitor cells, specific transcript analysis may reveal selective export defects

• Alternative splicing assessment:

  • RNA-sequencing of control versus THOC6-deficient neural cells reveals THOC6-dependent splicing alterations

  • Analysis of ribosomal RNA-depleted samples allows detection of pre-mRNA splicing defects

  • Research has identified 152 genes with significant alternative splicing events included or excluded in >10% of transcripts in THOC6-deficient neural progenitor cells

• Integration with intellectual disability gene databases:

  • Compare THOC6-dependent alternatively spliced genes with syndromic intellectual disability databases

  • Studies identified 185 alternatively spliced genes in THOC6^W100*/W100* and 105 alternatively spliced genes in THOC6^E188K/E188K neural progenitor cells that are known causative genes for syndromic intellectual disability

• Pathway enrichment analysis:

  • Biological pathway enrichment analysis of mis-spliced genes in THOC6-affected cells has revealed enrichment for functions in RNA splicing, cell projection organization, membrane trafficking, organelle organization, mitosis cell cycle, and DNA damage response

How can THOC6 antibodies be utilized to evaluate its potential as a glioma biomarker?

Recent research has identified THOC6 as a potential diagnostic and prognostic biomarker for glioma :

What controls are essential when using THOC6 antibody for oncology research?

For robust oncology research using THOC6 antibodies:

• Tissue controls:

  • Positive controls: Glioblastoma tissue known to express high THOC6 levels

  • Negative controls: Normal brain tissue with low THOC6 expression

  • Gradient controls: Lower-grade gliomas with intermediate expression levels

• Cell line controls:

  • High THOC6-expressing lines: Glioblastoma cell lines

  • Low THOC6-expressing lines: Normal human astrocytes or neural progenitor cells

  • Experimentally manipulated lines: THOC6-knockdown or overexpressing variants of the same cell line

• Antibody controls:

  • Isotype control: Rabbit IgG at matching concentration

  • No primary antibody control: Secondary antibody only

  • Peptide competition: Pre-incubation with immunizing peptide

• Expression validation:

  • Orthogonal methods: Validate protein expression findings with mRNA expression data

  • Multiple antibodies: Use different antibodies targeting distinct THOC6 epitopes

How can I optimize detection of THOC6 in pluripotent and neural lineage cells?

Neural and pluripotent stem cells require specific optimization strategies:

• Cell type-specific considerations:

  • Human pluripotent stem cells (hPSCs) and neural progenitor cells (NPCs) have been successfully used to study THOC6

  • SOX1-positive neuroprogenitor cells derived from human iPSCs show distinct THOC6 expression patterns compared to undifferentiated cells

• Sample preparation:

  • For Western blotting, load approximately 0.2 mg/mL of total protein lysate

  • For immunofluorescence, optimize fixation methods (4% paraformaldehyde works well for nuclear proteins)

  • Consider cell permeabilization optimization (0.1-0.5% Triton X-100)

• Co-staining markers:

  • Use SOX1 as a validated marker for neuroprogenitor identification

  • SOX1 is detectable at approximately 50 kDa and maintains neural cells in an undifferentiated state within developing CNS

  • Co-staining with PAX6 can help identify neuroepithelial regions

• Detection system considerations:

  • For Western blot analysis of neural samples, reducing conditions and 12-230 kDa separation systems have been successfully employed

  • For fluorescence microscopy, nuclear counterstains help delineate THOC6 nuclear localization

What approaches can be used to investigate THOC6's interaction with other TREX components?

To study THOC6's role in TREX complex formation and function:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use THOC6 antibody to immunoprecipitate the protein and associated complexes

  • Western blot for known TREX components (THOC5, ALYREF)

  • Research has shown that biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5

• Proximity ligation assay (PLA):

  • Enables in situ detection of protein-protein interactions with high sensitivity

  • Useful for detecting THOC6 interactions with other TREX components in intact cells

  • Provides spatial information about where interactions occur within the cell

• Analyzing TREX tetramer formation:

  • Size-exclusion chromatography followed by Western blotting can detect shifts in complex size

  • Native PAGE analysis can preserve intact complexes

  • Sucrose gradient ultracentrifugation can separate complexes by size

• Functional validation approaches:

  • RNA immunoprecipitation (RIP) can identify RNAs associated with THOC6-containing complexes

  • CLIP-seq (Crosslinking immunoprecipitation) can identify direct RNA binding sites

  • Rescue experiments with wildtype vs. mutant THOC6 can validate interaction-dependent functions

What are common issues when using THOC6 antibody and how can they be resolved?

When working with THOC6 antibody, researchers may encounter several challenges:

• Weak or no signal in Western blot:

  • Optimize protein loading (25-50 μg per lane recommended)

  • Try longer exposure times (successful detection reported with 180s exposure)

  • Verify antibody dilution (1:500-1:2000 range is recommended)

  • Ensure proper blocking (3% nonfat dry milk in TBST has been successful)

  • Consider different detection systems (ECL Basic Kit has been validated)

• High background in immunohistochemistry:

  • Optimize antibody dilution (start with 1:100)

  • Ensure proper antigen retrieval (TE buffer pH 9.0 recommended, with citrate buffer pH 6.0 as alternative)

  • Increase washing steps duration and number

  • Use more stringent blocking conditions

  • Consider tissue-specific autofluorescence quenching if applicable

• Inconsistent results across experiments:

  • Standardize protein extraction methods

  • Prepare fresh working dilutions before each experiment

  • Use consistent incubation times and temperatures

  • Include positive controls in each experiment (A431 cells, mouse ovary tissue, or Neuro-2a cells)

• Specificity concerns:

  • Validate with knockout/knockdown controls

  • Perform peptide competition assays

  • Compare results with alternative THOC6 antibodies

  • Consider species-specific optimization

How can I design experiments to study THOC6-dependent splicing alterations?

To investigate THOC6's role in RNA splicing:

• Experimental design:

  • Compare control samples with THOC6-deficient samples (knockout, knockdown, or patient-derived cells with biallelic THOC6 variants)

  • Include heterozygous controls where possible to assess gene dosage effects

  • Consider developmental timing effects, especially in neural models

• RNA extraction and quality control:

  • Use methods that preserve RNA integrity (RIN score >8)

  • For splicing studies, include ribosomal RNA depletion rather than poly(A) selection

  • Consider subcellular fractionation to separate nuclear and cytoplasmic RNA pools

• RNA-seq analysis workflow:

  • Use sufficient sequencing depth (>50M paired-end reads recommended)

  • Apply splicing-aware analysis tools (e.g., rMATS, MAJIQ, LeafCutter)

  • Focus on percent spliced in (PSI) metrics for each alternative splicing event

  • Research has identified significant alternative splicing events in >10% of transcripts in THOC6-deficient neural progenitor cells

• Validation approaches:

  • RT-PCR validation of selected splicing events

  • Mini-gene splicing reporters for mechanistic studies

  • RNA-protein interaction studies (RIP, CLIP) to assess direct effects

  • Rescue experiments with wildtype THOC6 expression

• Functional classification:

  • Pathway enrichment analysis of mis-spliced genes

  • Integration with intellectual disability gene databases

  • Previous research found enrichment for functions in RNA splicing, cell projection organization, membrane trafficking, organelle organization, mitosis cell cycle, and DNA damage response