OCT6 Antibody

What is OCT6 and why is it significant in research?

OCT6, also designated as POU3F1 and OTF6, is a member of the POU transcription factor family belonging to the Class-3 subfamily. It functions as a transcription factor that specifically binds to the octamer motif sequence (5'-ATTTGCAT-3') in DNA. OCT6 has demonstrated critical roles in early embryogenesis and neurogenesis, making it an important research target in developmental biology and neuroscience .

OCT6 exhibits dual regulatory capabilities in gene expression—it can function as a transcriptional activator when binding cooperatively with SOX4, SOX11, or SOX12 to gene promoters, and conversely, it can act as a transcriptional repressor of myelin-specific genes . This functional versatility makes OCT6 antibodies valuable tools for investigating transcriptional regulation mechanisms in various developmental and pathological contexts.

What applications are OCT6 antibodies validated for?

OCT6 antibodies have been validated for multiple experimental applications as demonstrated in the table below:

For optimal results, researchers should titrate the antibody concentration in each specific experimental system, as the optimal concentration may be sample-dependent .

How should OCT6 antibodies be stored and handled for maximum stability?

OCT6 antibodies should be stored at -20°C where they remain stable for one year after shipment. The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . According to manufacturer guidelines, aliquoting is unnecessary for -20°C storage. Some preparations (particularly 20μl sizes) may contain 0.1% BSA as a stabilizer .

For routine handling, avoid repeated freeze-thaw cycles which can compromise antibody performance. When working with the antibody, maintain cold chain practices and return to -20°C promptly after use to preserve functionality.

What is the recommended protocol for Western blot analysis using OCT6 antibodies?

For Western blot analysis with OCT6 antibodies, follow this optimized methodology based on validated protocols:

• Sample preparation: Lyse cells in RIPA buffer containing protease inhibitors

• Protein quantification: Determine concentration using Bradford or BCA assay

• SDS-PAGE: Load 20-50 μg of protein per lane on a 10-12% gel

• Transfer: Transfer proteins to PVDF membrane at 100V for 1 hour

• Blocking: Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute OCT6 antibody at 1:500-1:1000 in blocking solution and incubate overnight at 4°C

• Washing: Wash membrane 3× with TBST, 5 minutes each

• Secondary antibody: Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Washing: Wash membrane 3× with TBST, 5 minutes each

• Detection: Apply ECL substrate and image

• Analysis: The expected molecular weight of OCT6 is approximately 46 kDa

This protocol has been validated for detecting OCT6 in human, mouse, and rat samples, with positive detection confirmed in HEK-293 cells .

How can I generate and validate custom OCT6 antisera for specialized research applications?

For researchers requiring custom OCT6 antisera with specific epitope targeting, the following validated methodology can be employed:

• Clone a selected fragment of the OCT6 protein (e.g., amino acids 1-196) into a bacterial expression vector such as pQE9

• Express the His6-tagged Oct-6 fragment in bacteria using IPTG induction

• Lyse bacterial cells by sonication in 6M urea in PBS

• Purify the His6-tagged OCT6 peptide using Ni-NTA agarose beads:

  • Add cell lysate to 0.8 mM imidazole

  • Add 300 μl Ni-NTA agarose beads slurry

  • Incubate overnight to allow peptide binding

  • Elute peptide with 6M urea in PBS containing 80 mM imidazole

• Verify purity (>95%) via Coomassie-stained SDS-PAGE

• Immunize rabbits with four sequential injections at 4-week intervals using 0.5-1.0 mg OCT6 peptide resuspended in incomplete Freund's adjuvant

• Validate antisera specificity through Western blotting, immunohistochemistry, and electrophoretic mobility shift experiments

This approach has been successfully implemented for generating OCT6 antisera used in schizophrenia research examining OCT6 expression in temporal and frontal lobe specimens .

What are the optimal conditions for immunofluorescence staining with OCT6 antibodies?

For optimal immunofluorescence detection of OCT6, researchers should follow this validated protocol:

• Cell preparation: Culture cells on coverslips or chamber slides to 70-80% confluence

• Fixation: Fix with 4% paraformaldehyde (PFA) for 15 minutes at room temperature

• Permeabilization: Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Blocking: Block with 5% normal serum (from the species of secondary antibody) in PBS for 1 hour

• Primary antibody: Apply OCT6 antibody at a concentration of 4 μg/ml and incubate overnight at 4°C

• Washing: Wash 3× with PBS, 5 minutes each

• Secondary antibody: Apply fluorochrome-conjugated secondary antibody and incubate for 1 hour at room temperature in the dark

• Washing: Wash 3× with PBS, 5 minutes each

• Nuclear counterstain: Apply DAPI (1 μg/ml) for 5 minutes

• Mounting: Mount with anti-fade mounting medium

• Imaging: Analyze using confocal or fluorescence microscopy

This protocol has been successfully employed for OCT6 detection in various cell types, including SK-MEL-30 cells, demonstrating clear nuclear localization consistent with its function as a transcription factor .

How can OCT6 antibodies be used to investigate cellular reprogramming and pluripotency?

Recent research has revealed that OCT6 plays a significant role in maintaining pluripotency and inhibiting differentiation in induced pluripotent stem cells (iPSCs). To investigate OCT6's function in pluripotency maintenance, researchers can implement the following methodological approach:

• Generate stable cell lines overexpressing OCT6 using lentiviral vectors (e.g., PCDH-Teton-3×Flag-OCT6-Puro) with appropriate controls (empty vector)

• Verify OCT6 overexpression through:

  • qRT-PCR for mRNA expression

  • Western blotting for protein expression

  • Immunofluorescence for subcellular localization

• Assess pluripotency under differentiation conditions by:

  • Colony morphology analysis

  • Expression of pluripotency markers

  • Analysis of differentiation markers

• Perform RNA-seq to identify genes and pathways regulated by OCT6

• Validate key findings through qRT-PCR, Western blotting, and immunofluorescence

• Manipulate signaling pathways (e.g., ERK and PI3K-AKT) using small molecule compounds to further elucidate OCT6's mechanism of action

This approach has demonstrated that iPSCs overexpressing OCT6 maintain colony morphology and pluripotency markers under differentiation conditions, suggesting OCT6's role in resisting differentiation signals .

What strategies should be employed when investigating OCT6 expression in neurological disorders?

When investigating OCT6 expression in neurological disorders such as schizophrenia, researchers should consider the following methodological approach:

• Tissue acquisition: Obtain properly matched case-control specimens with attention to:

  • Postmortem interval

  • Age and sex matching

  • Medication history

  • Region-specific sampling (e.g., temporal and frontal lobes)

• Immunohistochemical analysis:

  • Use validated OCT6 antibodies with known specificity

  • Include positive and negative controls

  • Employ antigen retrieval methods if necessary

  • Quantify expression using standardized scoring methods

• Western blot confirmation:

  • Use freshly frozen tissue samples

  • Include loading controls

  • Quantify signal intensity relative to controls

• Correlative analysis:

  • Associate OCT6 expression patterns with clinical parameters

  • Compare with other disease-related markers

  • Consider regional differences in expression

Research has demonstrated that OCT6 is widely expressed in the temporal and frontal lobes of schizophrenic specimens while being essentially undetectable in matched control samples, suggesting its potential role as a disease marker or in pathophysiological mechanisms .

How can researchers analyze contradictory data in OCT6 functional studies across different cell types?

When faced with contradictory data regarding OCT6 function across different cell types or experimental systems, researchers should implement the following analytical approach:

• Perform comprehensive literature review to identify:

  • Cell type-specific functions

  • Context-dependent interactions with co-factors (e.g., SOX proteins)

  • Species-specific differences in OCT6 function

• Design comparative experiments:

  • Use identical antibody lots and dilutions across experiments

  • Implement parallel protocols in different cell types

  • Verify antibody specificity in each system

• Investigate protein-protein interactions:

  • Perform co-immunoprecipitation to identify cell-specific binding partners

  • Analyze post-translational modifications affecting OCT6 function

  • Consider chromatin immunoprecipitation (ChIP) to identify differential binding sites

• Conduct functional validation:

  • Use knockdown/knockout approaches to confirm specificity of observed effects

  • Employ rescue experiments with wild-type and mutant OCT6

  • Consider isoform-specific effects

This systematic approach can help resolve apparent contradictions, such as OCT6's dual role as a transcriptional activator (when cooperating with SOX factors) and a transcriptional repressor (of myelin-specific genes), by identifying the context-specific co-factors and signaling environments that dictate its function .

What are common issues encountered with OCT6 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with OCT6 antibodies. Here are evidence-based solutions for common problems:

Additionally, for tissue-specific detection issues, researchers should optimize antigen retrieval methods and fixation conditions based on the specific tissue being examined.

How should researchers optimize OCT6 antibody conditions for novel experimental systems?

When adapting OCT6 antibodies to novel experimental systems (new cell types, tissues, or methodologies), researchers should follow this systematic optimization approach:

• Initial validation in established positive control systems:

  • Confirm antibody functionality in HEK-293 cells for Western blot applications

  • Verify expected molecular weight (46 kDa) and specificity

• Antibody titration in the new system:

  • Perform a broad dilution series (e.g., 1:100, 1:500, 1:1000, 1:2000)

  • Select optimal concentration balancing specific signal and background

  • Document optimal conditions for future reproducibility

• Protocol optimization:

  • For challenging tissues, test multiple fixation methods (PFA, methanol, acetone)

  • Evaluate different antigen retrieval approaches for IHC/IF

  • Optimize blocking conditions (concentration, duration, blocking agent)

• Cross-validation:

  • Confirm findings with alternative detection methods

  • When possible, validate with genetic approaches (siRNA knockdown)

  • Compare results with published data on OCT6 expression patterns

Researchers should remember that sample-dependent factors may necessitate system-specific optimization, as indicated in product guidelines .

How can OCT6 antibodies contribute to understanding pluripotency networks in stem cell research?

Recent findings demonstrate that OCT6 plays a critical role in pluripotency maintenance in induced pluripotent stem cells (iPSCs). Researchers can leverage OCT6 antibodies to investigate pluripotency networks through the following approaches:

• ChIP-seq analysis to identify:

  • Genome-wide OCT6 binding sites

  • Co-occupancy with other pluripotency factors

  • Dynamic binding patterns during differentiation

• Comparative proteomics to elucidate:

  • OCT6 interaction partners in pluripotent versus differentiating cells

  • Post-translational modifications regulating OCT6 activity

  • Species-specific differences in OCT6 complexes

• Single-cell analysis to reveal:

  • Heterogeneity in OCT6 expression within stem cell populations

  • Correlation between OCT6 levels and differentiation propensity

  • Temporal dynamics during fate decisions

Research has shown that OCT6 overexpression maintains colony morphology and pluripotency under differentiation conditions, suggesting it plays a role in stabilizing the pluripotent state. This finding opens avenues for investigating how OCT6 interacts with canonical pluripotency factors like OCT4, SOX2, and KLF4 to modulate stem cell fate decisions .

What methodological advances are enhancing OCT6 antibody applications in neurodevelopmental research?

Emerging methodological approaches are expanding the utility of OCT6 antibodies in neurodevelopmental research:

• Multiplexed imaging technologies:

  • Simultaneously detect OCT6 with multiple lineage markers

  • Apply cyclic immunofluorescence to analyze dozens of markers on the same sample

  • Implement tissue clearing methods for 3D visualization of OCT6 expression patterns

• Live-cell imaging applications:

  • Utilize fluorophore-conjugated antibody fragments for dynamic studies

  • Track OCT6 localization during neuronal differentiation in real-time

  • Correlate OCT6 dynamics with morphological changes and gene expression

• Single-cell omics integration:

  • Combine antibody-based sorting of OCT6+ cells with single-cell RNA-seq

  • Perform CUT&Tag for single-cell epigenomic profiling of OCT6 binding

  • Integrate proteomics and transcriptomics data to build comprehensive regulatory networks

These methodological advances are particularly valuable for investigating OCT6's role in neurogenesis and its potential involvement in neurodevelopmental disorders, as suggested by its aberrant expression in schizophrenia specimens .

How can computational approaches enhance the interpretation of OCT6 antibody-based experiments?

Advanced computational methods are increasingly important for maximizing insights from OCT6 antibody-based experiments:

• Machine learning for image analysis:

  • Automated quantification of OCT6 expression in heterogeneous tissues

  • Pattern recognition to identify subtle phenotypic changes in OCT6-manipulated cells

  • Deep learning to predict OCT6 regulatory networks from multi-omics data

• Network biology approaches:

  • Construct gene regulatory networks centered on OCT6

  • Identify statistically significant pathway enrichments from OCT6 perturbation experiments

  • Model OCT6 interactions with other transcription factors using binding motif analysis

• Cross-species comparative analysis:

  • Analyze conservation of OCT6 function across evolutionary distances

  • Compare OCT6 binding sites and regulatory targets between species

  • Identify conserved and divergent aspects of OCT6 biology

Implementation of these computational approaches can help researchers interpret complex datasets and generate testable hypotheses. For example, RNA-seq analysis of OCT6-overexpressing iPSCs has revealed gene expression patterns that maintain pluripotency, providing insights into the molecular mechanisms by which OCT6 inhibits differentiation .