hoxc11a Antibody

What is HOXC11A and why is it important in research?

HOXC11A is part of the HOX gene family of transcription factors that play critical roles in embryonic development and tissue differentiation. HOX genes are known to regulate morphogenesis and cell differentiation during development. In zebrafish specifically, HOXC11A is involved in developmental patterning .

Research on HOXC11/HOXC11A is critical because understanding its mechanisms can potentially lead to new therapeutic targets for cancer treatment and provide insights into developmental biology.

What species reactivity can I expect from commercially available HOXC11A antibodies?

Based on available information, commercial HOXC11A antibodies show varying species reactivity:

• Confirmed reactivity: Zebrafish (Danio rerio) for antibodies targeting the N-terminal region (AA 1-30)

• Predicted reactivity based on sequence homology: Human, Mouse, Rat, Cow, Dog, Guinea Pig, Horse, and Rabbit

When selecting an antibody for your research, it's essential to verify the specific reactivity claimed by manufacturers and consider validating the antibody in your experimental system before proceeding with full-scale experiments. The high predicted homology across species suggests conservation of the protein structure, particularly in the N-terminal region, but experimental validation remains necessary since predicted reactivity doesn't always translate to actual performance .

What applications are HOXC11A antibodies validated for?

Currently available HOXC11A antibodies are primarily validated for Western Blotting (WB) applications . The recommended dilution for Western Blotting is typically 1:1000, though this may vary between specific antibody products .

While the antibodies are primarily validated for WB, based on the research on HOXC11, these antibodies might potentially be useful in other applications such as:

• Immunohistochemistry (IHC) for tissue samples

• Chromatin Immunoprecipitation (ChIP) assays to study DNA-protein interactions

• Immunofluorescence to visualize protein localization

How should HOXC11A antibodies be stored and handled?

For optimal performance and longevity of HOXC11A antibodies, follow these storage and handling guidelines:

• Short-term storage (up to 1 week): Store at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles that can degrade antibody quality

• Format: Most HOXC11A antibodies are supplied as liquid in PBS buffer with 0.09% sodium azide and sometimes with 2% sucrose as a stabilizer

• Safety precaution: Be aware that many antibody preparations contain sodium azide, which is classified as a poisonous and hazardous substance that should be handled by trained staff only

Creating small aliquots upon receipt is particularly important to minimize freeze-thaw cycles, which can significantly impact antibody performance. Each freeze-thaw cycle can reduce antibody activity, potentially leading to inconsistent experimental results .

How can I optimize Western Blot protocols for HOXC11A detection?

Optimizing Western Blot protocols for HOXC11A detection requires careful consideration of several factors:

Sample preparation:

• For cells: Lyse cells in RIPA buffer supplemented with protease inhibitors to prevent protein degradation

• For tissues: Homogenize tissues thoroughly in appropriate buffer and clarify lysates by centrifugation

Protein loading and separation:

• Load adequate protein (typically 20-40 μg per lane)

• Use 10-12% SDS-PAGE gels for optimal separation of HOXC11 proteins (molecular weight approximately 35-40 kDa)

Antibody incubation:

• Primary antibody dilution: Start with manufacturer's recommendation (typically 1:1000)

• Incubation time: Overnight at 4°C may yield better results than shorter incubations

• Blocking: Use 5% non-fat dry milk or BSA in TBST to reduce background

Detection optimization:

• If signal is weak: (1) Increase antibody concentration, (2) Extend incubation time, or (3) Use more sensitive detection systems

• If background is high: (1) Increase blocking time, (2) Add 0.1-0.3% Tween-20 to washing buffer, or (3) Dilute primary and secondary antibodies more

Based on research with HOXC11, it's worth noting that protein ubiquitination may affect detection, as HOXC11 has been shown to undergo ubiquitination that affects its stability . Consider using proteasome inhibitors in your lysate preparation if degradation is suspected.

What controls should I include when studying HOXC11A expression?

Including appropriate controls is critical for reliable interpretation of HOXC11A expression studies:

Positive controls:

• Cell lines or tissues with known HOXC11A expression (e.g., certain lung cancer cell lines for HOXC11)

• Recombinant HOXC11A protein (if available)

• For zebrafish studies, embryonic tissues during developmental stages when HOXC11A is known to be expressed

Negative controls:

• Cell lines with confirmed low or absent HOXC11A expression

• HOXC11A knockout cell lines (e.g., CRISPR/Cas9-generated) as ultimate negative controls

• Primary antibody omission control to assess secondary antibody specificity

Expression validation controls:

• Parallel qRT-PCR to correlate protein levels with mRNA expression

• Multiple antibodies targeting different epitopes of HOXC11A (if available)

• siRNA or shRNA knockdown of HOXC11A to confirm antibody specificity

As shown in HOXC11 research, different cell lines exhibit varying levels of expression, making them useful as comparative controls. For example, A549 and H23 lung cancer cell lines have been used as HOXC11 low-expressing cell lines, while PC9 has higher expression .

How can I investigate the role of HOXC11A in cancer progression?

Based on research with HOXC11 in lung adenocarcinoma, several methodological approaches can be applied to investigate HOXC11A's role in cancer:

Expression analysis:

• Compare HOXC11A expression between tumor tissues and adjacent normal tissues using IHC or Western blotting

• Correlate expression levels with clinical parameters and patient survival data

• Analyze public databases (e.g., TCGA, GEO) for HOXC11A expression patterns across cancer types

Functional studies:

• Generate stable overexpression and knockout cell lines using lentiviral vectors or CRISPR/Cas9

• Assess effects on:

  • Proliferation (MTT, BrdU incorporation)

  • Migration and invasion (Transwell assays)

  • Colony formation

  • Cell cycle progression (Flow cytometry)

In vivo models:

• Subcutaneous xenograft models to assess tumor growth

• Metastasis models (e.g., tail vein injection) to study metastatic potential

• Compare tumor volume, weight, and metastatic burden between HOXC11A-modified and control cells

Mechanistic investigations:

• ChIP assays to identify direct target genes

• RNA-seq to analyze transcriptome changes

• Co-immunoprecipitation to identify protein interaction partners

• Investigate potential regulatory pathways (e.g., NF-κB signaling which has been linked to HOXC11)

Research on HOXC11 has demonstrated that it regulates SPHK1 expression by directly binding to its promoter, suggesting that promoter binding studies are particularly valuable for understanding HOXC11A function .

What are the known signaling pathways and mechanisms through which HOXC11/HOXC11A functions?

Based on research with HOXC11, several important signaling mechanisms have been identified:

Transcriptional regulation:

• HOXC11 functions as a transcription factor that directly binds to promoter regions of target genes

• It specifically regulates sphingosine kinase 1 (SPHK1) expression by binding to its promoter region, which contributes to cancer progression

NF-κB pathway interactions:

• HOXC11 expression is regulated by IκB kinase α (IKKα), a pivotal kinase in NF-κB signaling

• This regulation is related to the ubiquitination of HOXC11, suggesting post-translational control mechanisms

Deubiquitination mechanisms:

• HOXC11 protein stability appears to be regulated by ubiquitination-deubiquitination balance

• USP8 (a deubiquitinating enzyme) has been shown to affect HOXC11 protein levels without direct binding, increasing HOXC11 when overexpressed

• USP8 expression can significantly reduce HOXC11 ubiquitination

Cell cycle regulation:

• HOXC11 overexpression accelerates cell cycle progression in cancer cells

• Conversely, HOXC11 knockout slows down cell cycle progression

Understanding these pathways provides potential targets for experimental intervention when studying HOXC11A functions and mechanisms.

What are common issues when using HOXC11A antibodies and how can they be resolved?

When working with HOXC11A antibodies, researchers may encounter several common issues:

Weak or no signal in Western blots:

• Potential causes: Insufficient protein, antibody degradation, low expression levels

• Solutions:

  • Increase protein loading (40-60 μg per lane)

  • Use fresh antibody aliquots

  • Enrich target protein (immunoprecipitation before WB)

  • Try enhanced chemiluminescence (ECL) systems with higher sensitivity

  • Extend exposure time gradually to detect weak signals

High background or non-specific bands:

• Potential causes: Insufficient blocking, antibody cross-reactivity, high antibody concentration

• Solutions:

  • Optimize blocking (try 5% BSA instead of milk, or vice versa)

  • Increase washing duration and number of washes

  • Further dilute primary and secondary antibodies

  • Try different antibody from another supplier targeting a different epitope

Inconsistent results between experiments:

• Potential causes: Antibody degradation, variable expression levels, technical variations

• Solutions:

  • Create single-use antibody aliquots to avoid freeze-thaw cycles

  • Standardize lysate preparation methods

  • Include consistent positive and negative controls

  • Normalize to loading controls and quantify bands using densitometry

Based on research with HOXC11, it's worth noting that protein expression may be influenced by regulatory mechanisms like ubiquitination , which could affect detection efficiency. Consider using proteasome inhibitors in your experimental design if protein degradation is suspected.

How can I validate HOXC11A antibody specificity for my experimental system?

Validating antibody specificity is crucial for reliable research outcomes:

Expression modulation experiments:

• Generate HOXC11A knockout controls (using CRISPR/Cas9 or similar technologies)

• Create HOXC11A overexpression systems

• Use siRNA or shRNA to knock down HOXC11A expression

• Confirm that antibody signal changes accordingly with these genetic manipulations

Peptide competition assays:

• Pre-incubate the antibody with excess immunizing peptide

• The specific signal should be abolished or significantly reduced while non-specific signals remain

Multi-technique validation:

• Correlate protein detection by Western blot with mRNA levels detected by qRT-PCR

• Use multiple antibodies targeting different epitopes of HOXC11A

• Employ immunoprecipitation followed by mass spectrometry to confirm identity

Species-specific considerations:

• For zebrafish studies, compare wild-type embryos with morpholino knockdown or mutant fish

• Include developmental timepoint controls when HOXC11A expression is known to change

Notably, in HOXC11 research, rescuing expression in knockout cell lines has been used to confirm antibody specificity and protein function, demonstrating that expression rescue reverses the phenotypic changes observed with knockout .

What considerations are important when designing experiments to study HOXC11A in developmental contexts?

When investigating HOXC11A in developmental contexts, particularly in zebrafish, consider these important experimental design factors:

Developmental timing:

• HOX genes exhibit temporal colinearity during development

• Carefully document and control the precise developmental stages of your samples

• Consider time-course experiments to capture dynamic expression changes

Spatial expression patterns:

• HOX genes show spatial colinearity along the anterior-posterior axis

• Use whole-mount immunohistochemistry or in situ hybridization to map expression domains

• Consider tissue-specific analyses rather than whole-embryo preparations when appropriate

Functional redundancy:

• HOX genes often show functional redundancy with paralogous genes

• Consider compound knockdown/knockout approaches targeting multiple paralogs

• Analyze compensation mechanisms by examining expression of related HOX genes after HOXC11A manipulation

Technical approaches:

• Use transgenic reporter lines to visualize HOXC11A expression domains in vivo

• Consider conditional knockout/knockdown systems to bypass early lethality

• Employ lineage tracing to identify HOXC11A-expressing cell populations and their descendants

Environmental factors:

• Control temperature precisely, as it affects developmental timing in poikilothermic organisms

• Standardize embryo density and culture conditions

• Consider that environmental stressors may alter HOX gene expression patterns

These considerations are based on general principles of HOX gene function and the specific information about HOXC11A antibodies being used in zebrafish research contexts .

How can I reconcile conflicting data about HOXC11/HOXC11A function across different experimental systems?

When faced with contradictory findings about HOXC11/HOXC11A function, consider these analytical approaches:

Context-dependent function analysis:

• HOX genes often have tissue-specific and developmental stage-specific functions

• Systematically compare experimental conditions (cell types, developmental stages, species)

• Consider that HOXC11 may function differently in normal versus cancer contexts

Technical variability assessment:

• Evaluate methodological differences between studies (antibodies used, knockdown efficiency, overexpression levels)

• Assess whether differences in mRNA versus protein analysis might explain discrepancies

• For example, in NSCLC research, conflicting results regarding HOXC11 function were attributed to differences in focusing on mRNA levels versus protein expression

Cofactor dependence:

• HOX proteins often require cofactors for functional specificity

• Investigate whether different cell types express different cofactors

• Consider analyzing expression of known HOX cofactors (e.g., MEIS, PBX proteins) in your experimental system

Reconciliation strategies:

• Perform side-by-side experiments in multiple systems using identical methods

• Use rescue experiments to confirm specificity of observed phenotypes

• Develop comprehensive models that incorporate context-dependent functions

For example, research has noted discrepancies in HOXC11's role in NSCLC, where some studies suggested tumor suppression while others indicated oncogenic functions. These were reconciled by considering differences in experimental approach and focusing on protein-level analysis rather than just mRNA expression .

What emerging techniques could enhance HOXC11A research beyond traditional antibody applications?

Several cutting-edge approaches offer new opportunities for HOXC11A research:

CRISPR-based technologies:

• CRISPR activation (CRISPRa) for endogenous gene upregulation

• CRISPR interference (CRISPRi) for targeted transcriptional repression

• CRISPR knock-in of tags (e.g., FLAG, HA) to circumvent antibody specificity issues

• Base editing to introduce specific mutations without double-strand breaks

Single-cell technologies:

• Single-cell RNA-seq to identify cell populations expressing HOXC11A

• Single-cell ATAC-seq to assess chromatin accessibility at HOXC11A target sites

• Spatial transcriptomics to map HOXC11A expression in tissue contexts

Live imaging approaches:

• Fluorescent protein tagging of endogenous HOXC11A using CRISPR

• Optogenetic control of HOXC11A expression or activity

• FRET-based sensors to study HOXC11A protein interactions in real-time

Structural and biophysical methods:

• Cryo-EM to determine HOXC11A protein complex structures

• ChIP-seq with HOXC11A antibodies to identify genome-wide binding sites

• HiChIP or Proximity Ligation-Assisted ChIP-seq to study long-range chromatin interactions mediated by HOXC11A

Computational approaches:

• AI-based prediction of HOXC11A binding sites and target genes

• Integrative multi-omics analysis to contextualize HOXC11A function

• Network analysis to identify HOXC11A-centered regulatory hubs

These approaches could help address limitations of traditional antibody-based methods while providing deeper mechanistic insights into HOXC11A function.

What are the key considerations when translating HOXC11A research findings to potential clinical applications?

When considering clinical translation of HOXC11A research findings, especially in cancer contexts, several important factors should be addressed:

Biomarker validation requirements:

• Establish robust detection methods with rigorous analytical validation

• Conduct retrospective analyses on diverse patient cohorts

• Determine sensitivity, specificity, and predictive value in clinical samples

• Compare with existing biomarkers through multivariate analysis

Target validation for therapeutic development:

• Confirm HOXC11A's causative (not merely correlative) role in disease

• Evaluate potential for compensatory mechanisms after HOXC11A inhibition

• Assess effects of HOXC11A modulation on normal tissues

• Identify synthetic lethal interactions that might enhance therapeutic specificity

Therapeutic approaches to consider:

• Direct targeting: Develop small molecules that disrupt HOXC11A-DNA binding

• Indirect targeting: Target downstream effectors like SPHK1 which is regulated by HOXC11

• Combinatorial approaches: Explore synergies between HOXC11A inhibition and standard therapies

• Patient stratification: Identify HOXC11A-high patients who might benefit from specific interventions

Translational challenges to address:

• Develop methods to effectively deliver HOXC11A-targeting therapeutics

• Establish proper timing of intervention in disease progression

• Determine potential resistance mechanisms

• Consider regulatory requirements for companion diagnostics if patient selection is needed

What are the most promising future research directions for HOXC11A studies?

Based on current understanding of HOXC11/HOXC11A, several promising research directions emerge:

Mechanistic studies:

• Further characterization of HOXC11A's transcriptional targets beyond SPHK1

• Deeper investigation of the IKKα-mediated regulation of HOXC11 and its ubiquitination dynamics

• Exploration of potential cofactors that may modify HOXC11A activity in different cellular contexts

• Comparative analysis of HOXC11A function across species to identify conserved versus divergent roles

Cancer biology applications:

• Expansion of HOXC11 studies to cancer types beyond lung adenocarcinoma

• Investigation of HOXC11 as a potential therapeutic target through development of specific inhibitors

• Exploration of HOXC11 as a predictive biomarker for treatment response

• Analysis of HOXC11's role in modulating tumor microenvironment and immune responses

Developmental biology:

• Detailed mapping of HOXC11A expression and function during zebrafish development

• Comparative analysis with mammalian models to identify evolutionarily conserved functions

• Investigation of epigenetic regulation of HOXC11A during development

• Exploration of HOXC11A's role in tissue regeneration contexts

Technological advances:

• Development of more specific and sensitive antibodies targeting different HOXC11A epitopes

• Application of spatial transcriptomics to map HOXC11A expression in complex tissues

• Use of organoid models to study HOXC11A function in three-dimensional tissue contexts

• Implementation of high-throughput screening approaches to identify modulators of HOXC11A activity

These directions build upon the foundation established by current research while extending into new territories with potential clinical and basic science implications.

How can researchers contribute to improving HOXC11A research tools and resources?

The advancement of HOXC11A research would benefit from collective efforts to improve available tools and resources:

Antibody validation and optimization:

• Conduct systematic cross-validation of commercially available antibodies

• Share detailed protocols and troubleshooting guides within the research community

• Develop and characterize new antibodies against different epitopes of HOXC11A

• Create monoclonal antibodies with enhanced specificity for various applications

Genetic resources development:

• Generate and share CRISPR knockout cell lines and animal models

• Develop conditional/inducible expression systems for temporal control

• Create fluorescent reporter lines for live imaging studies

• Share validated siRNA/shRNA sequences that effectively target HOXC11A

Data sharing and integration:

• Contribute to public databases with expression data across tissues and disease states

• Develop standardized analysis pipelines for HOXC11A expression in RNA-seq datasets

• Create accessible repositories of ChIP-seq data revealing HOXC11A binding sites

• Establish consortia-based approaches for integrative multi-omics analysis

Method standardization:

• Establish consensus protocols for HOXC11A detection in different sample types

• Develop quantitative standards for comparing HOXC11A expression levels across studies

• Create reference materials for antibody validation

• Publish negative results to help others avoid unsuccessful approaches