IRX4 Antibody

What is IRX4 and why is it significant in developmental biology research?

IRX4 (Iroquois Homeobox Protein 4) is a ventricle-specific transcription factor that plays a crucial role in cardiac chamber specification and development. It has gained significance in developmental biology research as it marks multipotent, ventricular-specific progenitor cells that contribute exclusively to ventricular myocardium. IRX4-positive cells exhibit cardiovascular potency, with the ability to generate endothelial cells, smooth muscle cells, and specifically ventricular myocytes. The ventricle-specific expression pattern makes IRX4 an invaluable marker for studying heart chamber differentiation and cardiac lineage specification during embryonic development . Recent studies have also implicated IRX4 in cancer biology, particularly in prostate cancer progression, suggesting its role extends beyond developmental contexts .

What experimental applications are IRX4 antibodies most commonly used for?

IRX4 antibodies are primarily utilized in three main experimental applications:

• Western Blotting (WB): For detecting and quantifying IRX4 protein expression in tissue or cell lysates, with recommended dilutions typically ranging from 1:500-1:2000 .

• Immunohistochemistry (IHC): For visualizing the spatial distribution of IRX4 in tissue sections, particularly useful for studying cardiac development and determining ventricle-specific expression patterns. Recommended dilutions generally range from 1:40-1:200 .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of IRX4 protein in solution, with higher dilutions typically used (1:5000-1:10000) .

Additionally, some specialized IRX4 antibodies have been validated for immunofluorescence (IF) applications, which are particularly valuable for co-localization studies with other cardiac markers .

What species reactivity should be considered when selecting an IRX4 antibody?

When selecting an IRX4 antibody, researchers should consider their experimental model and the cross-reactivity profile of available antibodies. Based on current commercial offerings, IRX4 antibodies demonstrate varied species reactivity profiles:

For developmental studies spanning multiple model organisms, selecting antibodies with broader species reactivity can facilitate comparative analyses. For human-specific applications such as cancer research, antibodies with exclusive human reactivity might be preferred for greater specificity .

What are the optimal protocols for using IRX4 antibodies in Western Blotting?

For optimal Western Blotting results with IRX4 antibodies, the following methodological considerations are recommended:

• Sample Preparation:

  • For cardiac tissue/cells: Lyse samples in RIPA buffer containing protease inhibitors

  • For embryonic tissues: Consider using gentler lysis conditions to preserve protein integrity

  • Load 20-40μg of total protein per lane

• Electrophoresis and Transfer:

  • Use 10-12% SDS-PAGE gels (IRX4 molecular weight: ~50-55 kDa)

  • Transfer to PVDF membrane (preferred over nitrocellulose for this application)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Antibody Incubation:

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

  • Dilute primary antibody 1:500-1:2000 in blocking solution

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3×10 minutes with TBST

  • Incubate with appropriate secondary antibody (typically anti-rabbit HRP at 1:5000-1:10000)

  • Wash 3×10 minutes with TBST

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Optimize exposure time based on signal intensity

For developmental studies tracking IRX4 expression during differentiation, consider running time-course experiments similar to those described in the literature where IRX4 expression was first detected at day 4.5 of embryoid body differentiation .

How should IRX4 antibodies be optimized for immunohistochemistry of cardiac tissues?

For effective immunohistochemical detection of IRX4 in cardiac tissues, the following protocol optimizations are recommended:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • For embryonic tissues, reduce fixation time to 6-12 hours to prevent over-fixation

  • Process and embed in paraffin, cutting sections at 5-7μm thickness

• Antigen Retrieval (critical step):

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0)

  • Boil for 20 minutes, then cool slowly to room temperature

• Antibody Application:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol

  • Block non-specific binding with 5-10% normal serum matching the secondary antibody host

  • Apply primary IRX4 antibody at 1:40-1:200 dilution

  • Incubate in a humidified chamber overnight at 4°C

  • For fluorescence detection, use appropriate fluorochrome-conjugated secondary antibodies

• Counterstaining and Controls:

  • For brightfield IHC, counterstain with hematoxylin

  • For IF, counterstain nuclei with DAPI

  • Include both positive controls (ventricle tissue) and negative controls (antibody omission and non-cardiac tissue)

For dual staining to identify Irx4+ ventricular progenitor cells, co-stain with markers such as Tbx5 or Islet1 to identify first and second heart field populations, respectively, as demonstrated in published protocols .

What strategies can be employed to validate the specificity of IRX4 antibodies?

Validating antibody specificity is critical for ensuring reliable research results. For IRX4 antibodies, employ these complementary validation approaches:

• Western Blot Analysis:

  • Confirm single band at expected molecular weight (~50-55 kDa)

  • Compare expression in tissues known to express IRX4 (ventricle) versus non-expressing tissues

  • Include IRX4 knockout/knockdown samples as negative controls

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots or IHC with blocked and unblocked antibody

  • Signal should be abolished or significantly reduced in the blocked sample

• Orthogonal Validation:

  • Compare protein detection with mRNA expression using RT-PCR or RNA-seq

  • Verify temporal expression patterns match known developmental timing (e.g., IRX4 upregulation between day 4.25-4.5 in embryoid body differentiation)

• Recombinant Expression System:

  • Overexpress tagged IRX4 in a cell line with low endogenous expression

  • Confirm antibody detection correlates with overexpression

  • Use tag-specific antibodies as controls

For reporter systems, validate co-expression of IRX4 and reporter genes by co-staining with antibodies to both IRX4 and the reporter, as demonstrated in the literature where researchers confirmed that reporter genes were restricted to Irx4+ cells .

How can IRX4 antibodies be utilized to investigate ventricular cardiomyocyte differentiation from stem cells?

IRX4 antibodies provide valuable tools for investigating ventricular specification during cardiomyocyte differentiation from stem cells:

• Temporal Profiling of Ventricular Specification:

  • Conduct time-course experiments during cardiac differentiation

  • Use flow cytometry with IRX4 antibodies to quantify emergence of ventricular progenitors

  • Correlate IRX4 expression with other cardiac markers (MLC2v, cTnT) to track ventricular commitment

• Purification of Ventricular Progenitors:

  • Employ IRX4 antibodies for fluorescence-activated cell sorting (FACS)

  • Combine with surface markers (Cxcr4, Pdgfr-alpha, Flk1, Flt1) for multi-parameter sorting

  • Culture sorted cells to assess proliferation capacity and differentiation potential

• Co-expression Analysis:

  • Perform dual immunostaining of IRX4 with first heart field marker Tbx5 and second heart field marker Islet1

  • Quantify co-expression to identify distinctive ventricular subpopulations

  • Track maintenance or loss of IRX4 expression in differentiated progeny (ventricular cardiomyocytes maintain IRX4 expression, while endothelial and smooth muscle cells lose it)

Research has demonstrated that Irx4+ ventricular progenitor cells (VPCs) are particularly prevalent between days 4.5-6 of embryoid body differentiation, and these cells exhibit cardiovascular potency. When differentiated, approximately 64.4% become cTnT+/Mlc2v+ ventricular cardiomyocytes, 23% develop into smooth muscle cells, and 12.5% become endothelial cells . This quantitative distribution provides a benchmark for evaluating the efficiency of ventricular differentiation protocols.

What role does IRX4 play in cancer research and how can antibodies facilitate these investigations?

Recent research has implicated IRX4 in cancer biology, particularly in relation to micropeptides and oncogenic signaling:

• Detection of IRX4-Derived Micropeptides:

  • Use highly sensitive IRX4 antibodies capable of detecting small peptide fragments

  • Apply SWATH-MS/MS-based proteomic approaches to identify micropeptides (miPEPs) generated from IRX clusters

  • Investigate the 17 identified miPEPs from IRX clusters implicated in prostate, breast, endometrial, and ovarian cancers

• Signaling Pathway Analysis:

  • Employ IRX4 antibodies to study the relationship between IRX4 expression and Wnt signaling dysregulation

  • Perform co-immunoprecipitation experiments to identify IRX4 protein interaction partners

  • Investigate how IRX4-derived micropeptides promote prostate cancer progression and chemoresistance

• Expression Analysis in Clinical Samples:

  • Use immunohistochemistry with IRX4 antibodies on tissue microarrays

  • Correlate expression levels with clinical outcomes and treatment response

  • Develop scoring systems based on staining intensity and distribution

The emerging role of IRX4 in cancer highlights its potential as a biomarker and therapeutic target. Genome-wide association studies have implicated Iroquois (IRX) gene clusters in cancer susceptibility, with IRX4-derived micropeptides potentially promoting prostate cancer progression through Wnt signaling dysregulation .

How can single-cell analysis be performed using IRX4 antibodies?

For single-cell analysis of IRX4 expression, researchers can employ several complementary approaches:

• Single-Cell Flow Cytometry:

  • Optimize cell preparation protocols to maintain viability and antigen integrity

  • Perform intracellular staining for IRX4 with permeabilization steps

  • Combine with surface markers for multiparametric analysis

  • Consider using conjugated IRX4 antibodies (FITC, Alexa Fluor 647, Alexa Fluor 680) for multicolor experiments

• Mass Cytometry (CyTOF):

  • Use metal-conjugated IRX4 antibodies

  • Combine with up to 40 additional markers for comprehensive phenotyping

  • Analyze data using dimensionality reduction techniques (t-SNE, UMAP)

• Single-Cell RNA-seq Integration:

  • Correlate protein expression (from antibody-based methods) with transcriptomic data

  • Validate IRX4 transcript-to-protein relationships

  • Identify potential post-transcriptional regulation

• Spatial Transcriptomics:

  • Combine IRX4 immunofluorescence with in situ hybridization techniques

  • Map spatial distribution of IRX4+ cells within tissue architecture

  • Correlate with neighboring cell populations and microenvironmental factors

Research has demonstrated that single Irx4+ ventricular progenitor cells exhibit cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro . This finding, achieved through single-cell analysis techniques, established the multipotent nature of these progenitors and their specific contribution to ventricular myocardium development.

What are common challenges when using IRX4 antibodies and how can they be addressed?

Researchers commonly encounter these challenges when working with IRX4 antibodies:

• Low Signal Intensity:

  • Cause: Insufficient antigen, suboptimal antibody dilution, or ineffective antigen retrieval

  • Solution: Increase antibody concentration, extend incubation time, optimize antigen retrieval (try different buffers: citrate pH 6.0 vs. EDTA pH 9.0), or use signal amplification systems

• High Background:

  • Cause: Excessive antibody concentration, insufficient blocking, or cross-reactivity

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional washing steps, or use more specific detection systems

• Inconsistent Results Between Experiments:

  • Cause: Variations in sample processing, antibody lots, or protocol execution

  • Solution: Standardize protocols, include positive controls in each experiment, and maintain consistent antibody lots for long-term studies

• Non-specific Bands in Western Blot:

  • Cause: Cross-reactivity with related proteins, degradation products, or post-translational modifications

  • Solution: Increase stringency of washing conditions, use monoclonal antibodies if available, or validate with peptide competition assays

• Epitope Masking in Fixed Tissues:

  • Cause: Over-fixation or protein-protein interactions blocking epitope access

  • Solution: Optimize fixation conditions, try alternative antigen retrieval methods, or consider using fresh-frozen sections

When working with IRX4 antibodies for ventricular progenitor cell identification, researchers have successfully addressed these challenges by implementing a reporter system that integrates luciferase, hygromycin resistance, and tdTomato reporter genes into the IRX4 locus, providing a complementary approach when antibody detection is challenging .

How can IRX4 antibody performance be optimized for detecting low abundance targets?

For detecting low abundance IRX4 or IRX4-derived peptides, consider these optimization strategies:

• Signal Amplification Systems:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry

  • Use highly sensitive chemiluminescent substrates for Western blotting

  • Consider biotin-streptavidin amplification systems with biotinylated IRX4 antibodies

• Sample Enrichment Techniques:

  • Perform subcellular fractionation to isolate nuclear fractions (where transcription factors typically localize)

  • Use immunoprecipitation to concentrate IRX4 protein before detection

  • Employ protein concentration methods for dilute samples

• Alternative Detection Methods:

  • Utilize proximity ligation assay (PLA) for detecting protein-protein interactions involving IRX4

  • Consider droplet digital PCR for absolute quantification of IRX4 transcripts as a complementary approach

  • Implement SWATH-MS/MS-based proteomic approaches for detecting IRX4-derived micropeptides

• Technical Modifications:

  • Extend primary antibody incubation time (up to 48-72 hours at 4°C)

  • Reduce washing stringency slightly (shorter washes or lower salt concentration)

  • Use fluorescently-conjugated IRX4 antibodies (e.g., Alexa Fluor 647, Alexa Fluor 680) for direct detection

For micropeptide research applications, where detection sensitivity is particularly critical, researchers have successfully identified 17 miPEPs generated from IRX clusters using specialized mass spectrometry techniques that can be complemented with targeted antibody-based validation .

What control samples are essential when establishing IRX4 antibody protocols?

Proper experimental controls are crucial for validating IRX4 antibody specificity and performance:

• Positive Controls:

  • Tissue Controls: Ventricular myocardium (high endogenous expression)

  • Cell Line Controls: Cardiomyocyte cell lines with confirmed IRX4 expression

  • Overexpression Controls: Cells transfected with IRX4 expression constructs

  • Developmental Controls: Day 7-10 embryoid bodies from cardiac differentiation protocols

• Negative Controls:

  • Tissue Controls: Non-cardiac tissues (e.g., liver, spleen)

  • Antibody Controls: Primary antibody omission, isotype controls

  • Genetic Controls: IRX4 knockout or knockdown samples

  • Peptide Competition: Antibody pre-incubated with immunizing peptide

• Technical Controls:

  • Loading Controls: Housekeeping proteins (β-actin, GAPDH) for Western blots

  • Staining Controls: Nuclear counterstains (DAPI, Hoechst) for microscopy

  • Processing Controls: Samples processed identically except for the variable being tested

• Differentiation Stage Controls:

  • Time course samples from different stages of cardiac differentiation

  • Multi-marker analysis to correlate IRX4 expression with known stage-specific markers

Research has established that Irx4+ cells should co-express specific markers (Cxcr4, Pdgfr-alpha, Flk1, Flt1) on the cell surface, and these can serve as validation markers when establishing new IRX4 antibody protocols .

How might IRX4 antibodies contribute to regenerative medicine applications?

IRX4 antibodies have significant potential in advancing regenerative medicine approaches for cardiac repair:

• Ventricular-Specific Cell Therapy:

  • Use IRX4 antibodies to isolate and purify ventricular progenitors for transplantation

  • Develop sorting strategies based on IRX4 expression for clinical-grade cell preparation

  • Monitor engraftment and differentiation of transplanted cells in recipient tissue

• Bioengineered Cardiac Tissues:

  • Employ IRX4 antibodies to characterize the ventricular specification of cells in engineered tissues

  • Validate chamber-specific organization in bioprinted cardiac constructs

  • Assess maturation state of ventricular cardiomyocytes in tissue engineered products

• Disease Modeling:

  • Apply IRX4 antibodies to identify chamber-specific defects in patient-derived cardiac organoids

  • Track ventricular development in congenital heart disease models

  • Evaluate chamber-specific drug responses in personalized medicine applications

Research has demonstrated that Irx4+ ventricular progenitor cells injected into the cardiac crescent of developing mouse embryos specifically contribute to the nascent ventricle, highlighting their potential for targeted regenerative applications . This ventricular specificity could be leveraged to develop more precise approaches for treating ventricle-specific cardiac disorders.

What is the relationship between IRX4 expression and cancer progression mechanisms?

Recent research has begun to uncover complex relationships between IRX4 expression and cancer biology:

• IRX4-Derived Micropeptides in Oncogenesis:

  • IRX4-derived micropeptides (small peptides encoded by short open reading frames) have been identified in multiple cancer types

  • These micropeptides appear to promote prostate cancer progression and chemoresistance

  • The mechanism involves dysregulation of Wnt signaling pathways

• Cancer Type Specificity:

  • IRX4 involvement has been documented in prostate, breast, endometrial, and ovarian cancers

  • Different IRX cluster-derived micropeptides may have tissue-specific functions

  • Genome-wide association studies have implicated IRX gene clusters in cancer susceptibility

• Potential Therapeutic Implications:

  • Targeting IRX4 or its derived micropeptides might represent a novel therapeutic approach

  • Antibodies against IRX4 could help identify patients likely to benefit from Wnt pathway inhibitors

  • Monitoring IRX4 expression might predict chemotherapy resistance

This emerging area requires additional research to fully elucidate the mechanisms by which IRX4-derived micropeptides influence cancer progression and therapy resistance. Antibody-based detection of these micropeptides will be essential for advancing this field and developing potential therapeutic interventions .