MICAL2 Antibody

What is MICAL2 and what cellular functions does it regulate?

MICAL2 (Microtubule Associated Monooxygenase, Calponin and LIM Domain Containing 2) is an atypical multidomain flavoenzyme with diverse cellular functions. It functions as a flavin monooxygenase enzyme that induces actin depolymerization and indirectly promotes serum response factor transcription by modulating the availability of serum response factor coactivators such as myocardin-related transcription factors (MRTF-A and MRTF-B) . MICAL2 has a calculated molecular weight of 127 kDa, though it is often observed at 95 kDa and 112 kDa in experimental contexts . It plays critical roles in cytoskeletal regulation, cell migration, and has been implicated in various cellular processes including myogenic differentiation and cancer progression . Recent research has demonstrated its importance in actin dynamics, which affects multiple downstream signaling pathways including KRAS signaling and epithelial-mesenchymal transition .

What are the recommended applications for MICAL2 antibody in different experimental settings?

MICAL2 antibody has been validated for multiple applications, with specific protocols optimized for each technique:

When designing experiments, researchers should note that the optimal dilution is sample-dependent and should be titrated in each testing system to obtain optimal results . Published applications have successfully used this antibody in knockout/knockdown studies, providing additional validation of specificity .

How should tissue samples be prepared for optimal MICAL2 detection in immunohistochemistry?

For immunohistochemistry applications, proper sample preparation is critical for sensitive and specific MICAL2 detection. Antigen retrieval methods significantly impact staining quality. For MICAL2 antibody (such as 13965-1-AP), the recommended protocol includes:

• Primary fixation with 4% paraformaldehyde for 15 minutes at room temperature

• Three PBS washes

• Permeabilization with 1% Bovine Serum Albumin (BSA) + 0.2% or 0.5% triton for 30-45 minutes at room temperature

• Blocking with 10% donkey serum for 30 minutes at room temperature

• Overnight incubation with primary antibody at 4°C using the appropriate dilution

For paraffin-embedded tissues, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may serve as an alternative . For frozen sections, the protocol used in murine model studies involves cutting transversally in 7 μm sections using a cryostat machine followed by standard immunofluorescence procedures .

How can researchers validate MICAL2 antibody specificity in their experimental systems?

Establishing antibody specificity is crucial for reliable research. For MICAL2 antibody, a comprehensive validation approach should include:

• Genetic validation: Using MICAL2 knockout or knockdown models as negative controls. Published research has successfully employed this approach in multiple studies .

• Molecular weight verification: Comparing observed bands with expected molecular weights. MICAL2 has a calculated molecular weight of 127 kDa but is typically observed at 95 kDa and 112 kDa in Western blots .

• Cross-reactivity testing: Verifying reactivity across species. The MICAL2 antibody (13965-1-AP) shows tested reactivity with human, mouse, and rat samples .

• Positive control selection: Using cell lines with confirmed MICAL2 expression, such as DU 145 cells, PC-3 cells, U-251 cells, or U-87 MG cells for Western blotting, and HepG2 cells for immunofluorescence .

• Peptide competition assay: Pre-incubating the antibody with its immunizing peptide (such as MICAL2 fusion protein Ag4950) to confirm binding specificity .

Each validation step should be thoroughly documented, and results compared with published literature to confirm consistency in detection patterns.

What methodological approaches can overcome challenges in detecting different MICAL2 isoforms?

Detecting specific MICAL2 isoforms presents technical challenges that require specialized approaches:

• Gel percentage optimization: Use gradient gels (4-12%) to achieve better separation of high molecular weight proteins and improve resolution between isoforms.

• Sample preparation considerations: Different extraction methods may preferentially isolate certain isoforms. For comprehensive isoform detection, compare multiple lysis buffers (RIPA, NP-40, and Triton-based buffers) to determine optimal extraction conditions.

• Antibody selection: The epitope location within the MICAL2 protein influences which isoforms will be detected. The antibody (13965-1-AP) targets a fusion protein of MICAL2 (Ag4950) , but researchers should confirm whether this epitope is present in all isoforms of interest.

• Combined detection methods: Complement protein detection with mRNA analysis (RT-PCR with isoform-specific primers) to validate isoform expression patterns at the transcriptional level.

• Mass spectrometry validation: For definitive isoform identification, immunoprecipitate MICAL2 and analyze by mass spectrometry to confirm the presence of isoform-specific peptides.

When reporting MICAL2 detection, clearly specify which isoforms were observed based on molecular weight, as the observed weights (95 kDa and 112 kDa) differ from the calculated weight (127 kDa) .

What approaches should researchers use to investigate MICAL2's role in pancreatic cancer progression?

Recent research has identified MICAL2 as a critical factor in pancreatic ductal adenocarcinoma (PDAC) progression, necessitating specific experimental approaches:

• Expression analysis in clinical samples: Utilize tissue microarray and comprehensive bioinformatics analysis combining gene expression data with clinical information from multiple datasets to correlate MICAL2 expression with patient outcomes. This approach has revealed that MICAL2 is highly expressed in pancreatic cancer tissue and associated with poor prognosis .

• Super-enhancer landscape characterization: Recent studies identified MICAL2 as a super-enhancer-associated gene in human PDAC, suggesting its role as a potential disease driver. Researchers should incorporate epigenomic profiling methods like ChIP-seq to examine regulatory elements controlling MICAL2 expression .

• Functional studies using loss/gain-of-function models: Implement both knockdown and overexpression approaches in human and mouse PDAC cells to evaluate effects on:

  • ERK1/2 and AKT activation

  • KRAS signaling pathway

  • Macropinocytosis (which is affected by MICAL2's role in actin depolymerization)

  • Cell-cycle progression

  • In vitro migration and proliferation

• In vivo tumor models: Assess MICAL2's impact on tumor growth and metastasis using appropriate animal models, which have demonstrated that MICAL2 supports tumor growth and metastatic spread .

• Tumor microenvironment analysis: Investigate MICAL2's relationship with immunosuppressive features, including cancer-associated fibroblast infiltration, M2 macrophage presence, and CD8+ T cell reduction .

These methodologies collectively provide a comprehensive framework for understanding MICAL2's multifaceted roles in pancreatic cancer biology.

How does MICAL2 influence the tumor microenvironment and what methods best characterize these interactions?

MICAL2 has emerged as a significant modulator of the tumor microenvironment, particularly in pancreatic cancer. To effectively study these interactions, researchers should employ these methodological approaches:

• Single-cell RNA sequencing: This technique helps identify the specific cell populations expressing MICAL2 within the tumor microenvironment. Research has shown that MICAL2 is mainly expressed in fibroblasts of pancreatic cancer .

• Spatial transcriptomics and multiplex immunofluorescence: These approaches map the relative positions of MICAL2-expressing cells in relation to other cell types in the tumor microenvironment, particularly immune cells.

• Immune cell infiltration analysis: Quantitative assessment of various immune cell populations has revealed that higher MICAL2 expression correlates with:

  • Increased cancer-associated fibroblast infiltration

  • Enhanced M2 macrophage presence

  • Reduced CD8+ T cell infiltration

These changes collectively contribute to an immunosuppressive microenvironment.

• Functional pathway analysis: Gene set enrichment and pathway analyses have demonstrated that MICAL2 is closely associated with:

  • Epithelial-mesenchymal transformation

  • Extracellular matrix degradation

  • Inflammatory response modulation

• Co-culture systems: Implementing co-culture models of cancer cells with fibroblasts and immune cells allows for functional validation of MICAL2's role in intercellular communication and immune modulation.

Understanding these interactions provides potential targets for combined therapies that address both MICAL2-mediated tumor progression and immunosuppression .

What experimental design best elucidates MICAL2's role in epithelial-mesenchymal transition (EMT) in cancer cells?

To effectively investigate MICAL2's contribution to epithelial-mesenchymal transition in cancer progression, researchers should implement a multi-faceted experimental design:

• Transcriptional profiling: Perform RNA-seq analysis comparing MICAL2 knockdown and overexpression models to identify changes in EMT-related gene signatures. Recent research has demonstrated that MICAL2 upregulates EMT signaling pathways, contributing to tumor growth and metastasis .

• EMT marker assessment: Systematically analyze changes in:

  • Epithelial markers (E-cadherin, ZO-1)

  • Mesenchymal markers (N-cadherin, Vimentin)

  • EMT transcription factors (SNAIL, SLUG, ZEB1/2, TWIST)
    using Western blot, qPCR, and immunofluorescence techniques

• Actin cytoskeleton visualization: Since MICAL2 functions as a flavin monooxygenase that induces actin depolymerization , perform detailed actin cytoskeleton imaging using phalloidin staining coupled with high-resolution microscopy to capture cytoskeletal rearrangements associated with EMT.

• Migration and invasion assays: Implement real-time cell analysis systems, wound healing assays, and transwell invasion assays to quantify functional changes in cell motility and invasiveness resulting from MICAL2 modulation.

• MRTF-SRF pathway analysis: Investigate how MICAL2-mediated actin dynamics affect the nuclear translocation and activity of myocardin-related transcription factors (MRTF-A and MRTF-B), which are known MICAL2 downstream effectors . This can be accomplished through:

  • MRTF nuclear/cytoplasmic fractionation

  • SRF-responsive luciferase reporter assays

  • ChIP-seq to identify MRTF/SRF binding sites across the genome

• In vivo metastasis models: Utilize appropriate animal models to evaluate how MICAL2 manipulation affects metastatic spread, validating in vitro findings in physiologically relevant systems .

This comprehensive approach allows researchers to establish mechanistic links between MICAL2 activity, cytoskeletal reorganization, and the EMT program in cancer progression.

What are effective troubleshooting strategies for inconsistent MICAL2 detection in Western blotting?

When encountering variable MICAL2 detection in Western blotting, researchers should systematically evaluate and optimize these parameters:

• Sample preparation optimization:

  • Ensure complete protein extraction using appropriate lysis buffers with protease inhibitors

  • Standardize protein quantification methods for consistent loading

  • Consider phosphatase inhibitors if studying phosphorylated forms

• Gel electrophoresis conditions:

  • Optimize acrylamide percentage (8-10% recommended for MICAL2's observed molecular weights of 95 kDa and 112 kDa)

  • Adjust running time and voltage for optimal separation

  • Consider gradient gels for better resolution

• Transfer conditions:

  • MICAL2's size may require extended transfer times or semi-dry systems

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Antibody optimization:

  • Titrate antibody concentration within the recommended range (1:2000-1:10000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Optimize blocking conditions to reduce background

  • Test different secondary antibodies

• Detection system sensitivity:

  • Choose appropriate detection chemistry (standard ECL vs. enhanced sensitivity systems)

  • Adjust exposure times based on signal strength

  • Consider digital imaging systems with adjustable sensitivity

• Positive control inclusion:

  • Include validated positive controls such as DU 145 cells, PC-3 cells, U-251 cells, or U-87 MG cells which have confirmed MICAL2 expression

  • Consider using cell lines with MICAL2 overexpression as strong positive controls

• Normalization strategy:

  • Use α-TUBULIN as a validated housekeeping protein for normalization

  • Employ relative densitometry, normalizing MICAL2 bands against background and housekeeping protein

By systematically addressing these variables, researchers can achieve consistent and reproducible MICAL2 detection in Western blotting experiments.

How can researchers optimize MICAL2 antibody performance in immunohistochemistry of different tissue types?

Optimizing MICAL2 antibody performance across diverse tissue types requires careful protocol adjustment:

• Tissue-specific fixation and processing:

  • For frozen sections: The 7 μm thickness used in murine model studies provides optimal results

  • For FFPE samples: Standardize fixation time to prevent overfixation that may mask epitopes

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: Citrate buffer pH 6.0

  • Systematically compare heat-induced epitope retrieval methods (microwave, pressure cooker, water bath) for each tissue type

• Blocking strategy adjustment:

  • Standard approach: 10% donkey serum for 30 minutes at room temperature

  • For tissues with high background: Consider additional blocking with avidin/biotin blocking kit or mouse-on-mouse blocking for mouse tissues

• Antibody concentration titration:

  • Starting dilution range: 1:50-1:500

  • Generate a dilution series and evaluate signal-to-noise ratio for each tissue type

  • Adjust incubation time and temperature based on staining intensity

• Detection system selection:

  • For tissues with low MICAL2 expression: Consider amplification systems (TSA, polymer-based)

  • For multiplex staining: Implement spectral imaging and unmixing techniques

• Counterstain adaptation:

  • Adjust hematoxylin counterstaining time based on tissue density

  • Consider nuclear counterstains compatible with downstream digital analysis

• Validation in relevant tissues:

  • For cancer studies: Human prostate cancer tissue has been validated for IHC

  • For studies involving pancreatic cancer: Tissue microarray approaches have been successfully employed

• Sample-specific controls:

  • Include tissue-matched positive and negative controls

  • Consider gradient tissue blocks with known differential expression

This methodical approach enables researchers to establish reliable, tissue-optimized protocols for consistent MICAL2 detection across different experimental contexts.

What methodological approaches should be used to study MICAL2's role in myogenic differentiation?

Investigating MICAL2's function in myogenic differentiation requires specialized approaches targeting stem cell differentiation and muscle development:

• Developmental expression analysis:

  • Track MICAL2 expression during myogenic differentiation of adult and pluripotent stem cells (PSCs) toward skeletal, smooth, and cardiac muscle lineages

  • Implement time-course studies with regular sampling to capture dynamic expression changes

• Embryoid body (EB) cardiac differentiation protocol:

  • Generate EBs using the hanging drop method (500 cells per drop)

  • Culture in cardiac differentiation medium for 48 hours in hanging drops

  • Collect drops and grow in suspension in ultra-low attachment plates for 5 days

  • Transfer to gelatin-coated plates for adhesion culture

  • Observe for beating areas starting from day 9 of differentiation

• Differentiation medium composition:

  • G-MEM supplemented with 20% FBS

  • 1% penicillin/streptomycin solution

  • 2 mmol/L glutamine

  • 1 mmol/L sodium pyruvate

  • 1% NEAA

  • 1:500 β-mercaptoethanol

• MICAL2 manipulation strategies:

  • Implement gain-of-function (overexpression) and loss-of-function (siRNA, CRISPR) approaches

  • Use inducible expression systems to control MICAL2 levels at specific differentiation stages

• Muscle-specific marker assessment:

  • Track expression of early (MyoD, Myf5) and late (MHC, Troponin) myogenic markers

  • Implement immunofluorescence visualization using appropriate antibody dilutions and imaging systems

• Actin dynamics evaluation:

  • Assess actin polymerization state using fluorescent phalloidin staining

  • Quantify G-actin/F-actin ratios in response to MICAL2 manipulation

• Functional myogenic assays:

  • For cardiac lineage: Measure beating frequency and synchronicity

  • For skeletal muscle: Assess myotube formation, fusion index, and contractile properties

• In vivo validation:

  • Analyze MICAL2 expression in skeletal muscle and hearts of murine models

  • Implement tissue-specific knockout models to evaluate developmental consequences

These methodologies provide a comprehensive framework for elucidating MICAL2's role in the complex process of myogenic lineage commitment and differentiation.

How can MICAL2-MRTF-SRF signaling axis be effectively studied in experimental systems?

The MICAL2-MRTF-SRF signaling axis represents an important regulatory pathway affecting gene expression through actin dynamics. To comprehensively investigate this signaling axis:

• Actin dynamics quantification:

  • Measure G-actin/F-actin ratios following MICAL2 manipulation using biochemical fractionation

  • Implement live-cell imaging with fluorescent actin probes to visualize dynamic changes

  • Quantify actin depolymerization rates in response to modulated MICAL2 activity

• MRTF nuclear translocation assessment:

  • Track MRTF-A and MRTF-B subcellular localization using immunofluorescence

  • Implement biochemical fractionation to quantify nuclear/cytoplasmic MRTF ratios

  • Utilize live-cell imaging with fluorescently tagged MRTF constructs

• MRTF-SRF transcriptional activity measurement:

  • Deploy SRF-responsive luciferase reporter assays

  • Perform ChIP-seq analysis to identify genome-wide SRF binding sites affected by MICAL2

  • Conduct RNA-seq following MICAL2/MRTF manipulations to identify downstream transcriptional programs

• Comparative MRTF-A vs. MRTF-B analysis:

  • Implement isoform-specific knockdown and overexpression

  • Recent research has shown that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo, suggesting distinct functional roles

• Pathway integration analysis:

  • Investigate cross-talk with KRAS signaling, as MICAL2 has been shown to promote both ERK1/2 and AKT activation

  • Examine epithelial-mesenchymal transition signaling pathway connections

  • Assess effects on macropinocytosis, which is influenced by actin dynamics and KRAS signaling

• Functional outcome assessment:

  • Evaluate proliferation, migration, and cell-cycle progression following manipulation of MICAL2, MRTF-A, or MRTF-B

  • Compare in vitro findings with in vivo tumor growth and metastasis models

• Pharmacological intervention:

  • Test actin-targeting compounds or SRF inhibitors in combination with MICAL2 manipulation

  • Evaluate potential for therapeutic targeting of this pathway in disease models

This comprehensive approach allows for detailed characterization of the MICAL2-MRTF-SRF signaling axis and its functional implications in normal and pathological contexts.