MAB21L2 Antibody

What is MAB21L2 and why is it important in developmental biology?

MAB21L2 is a vertebrate member of the Male-abnormal 21 (mab-21) gene family with critical roles in embryonic development. It functions in key developmental processes including gastrulation, neural tube formation, and eye morphogenesis. Loss-of-function studies have demonstrated its considerable importance in proper development across vertebrate species . The protein is highly conserved evolutionarily, suggesting fundamental biological functions that have been maintained through natural selection. MAB21L2's significance lies in its role as a developmental regulator that interacts with essential signaling pathways, particularly the BMP signaling cascade through SMAD protein interactions .

What cellular functions and molecular interactions has MAB21L2 been shown to participate in?

MAB21L2 has been demonstrated to function as a BMP4 antagonist, specifically interacting with the BMP4 effector SMAD1. Biochemical studies have revealed that MAB21L2 immunoprecipitates with SMAD1 in vivo and binds both SMAD1 and the SMAD1-SMAD4 complex in vitro . Importantly, while the interaction with SMAD1 appears direct, MAB21L2 does not directly contact SMAD4 but rather interacts with it through SMAD1-mediated assembly .

Furthermore, MAB21L2 demonstrates RNA-binding capability, specifically binding to single-stranded RNA, although this function is lost in mutated forms of the protein . When targeted to heterologous promoters, MAB21L2 acts as a transcriptional repressor, suggesting a potential role in gene expression regulation . This collection of interactions positions MAB21L2 as a multifunctional protein involved in developmental signaling pathways and potentially in post-transcriptional regulation.

How should I select the appropriate MAB21L2 antibody for my research?

When selecting a MAB21L2 antibody, consider these factors based on your experimental needs:

• Target species compatibility: Confirm the antibody's reactivity with your experimental model organism. Available antibodies show reactivity with human and mouse MAB21L2 .

• Application compatibility: Verify the antibody's validation for your intended application. Current MAB21L2 antibodies are validated for:

  • ELISA (recommended dilution 1:2000-1:10000)

  • Western Blotting (recommended dilution 1:500-1:5000)

  • Immunofluorescence (recommended dilution 1:50-1:200)

• Epitope specificity: Some antibodies target specific amino acid regions, such as AA 1-300 or AA 185-219, which may affect detection of splice variants or mutated proteins .

• Antibody format: Consider whether you need a conjugated (FITC, HRP, Biotin) or unconjugated antibody based on your detection system .

• Clonality: Both polyclonal and monoclonal antibodies are available for MAB21L2. Polyclonals offer broader epitope recognition while monoclonals provide higher specificity .

Select based on these criteria and your experimental design rather than commercial considerations.

What are the optimal conditions for Western blot detection of MAB21L2?

For optimal Western blot detection of MAB21L2, follow this evidence-based protocol:

• Sample preparation:

  • For tissue samples: Homogenize mouse liver tissue (where MAB21L2 has been successfully detected) in RIPA buffer with protease inhibitors .

  • For cell cultures: Harvest cells (P19 cells work well) 48 hours after desired treatment (BMP4 treatment can enhance detection) .

• Electrophoresis and transfer:

  • Load 20-30 μg protein per lane on 10-12% SDS-PAGE

  • Transfer to PVDF membrane (nitrocellulose also acceptable)

• Antibody incubation:

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

  • Incubate with primary MAB21L2 antibody (optimal dilution 1:500-1:5000)

  • Wash 3x with TBST

  • Incubate with appropriate secondary antibody (e.g., goat anti-rabbit IgG at 1:50000 dilution)

• Detection considerations:

  • Expected molecular weight: ~40 kDa

  • Positive control: Mouse liver tissue is recommended

  • Signal enhancement: BMP4 treatment of cells can increase MAB21L2-SMAD1 interactions

This protocol is based on validated experimental procedures from published research and commercial antibody validation data.

How can I optimize immunofluorescence protocols for MAB21L2 detection?

For optimal immunofluorescence detection of MAB21L2, implement the following protocol:

• Sample preparation:

  • Fixation: 4% paraformaldehyde for 15 minutes (preserves protein-protein interactions)

  • Permeabilization: 0.1% Triton X-100 for 10 minutes (allows antibody access)

  • Blocking: 3% BSA in PBS for 1 hour (reduces non-specific binding)

• Antibody incubation:

  • Primary antibody: Apply MAB21L2 antibody at dilution 1:50-1:200

  • Incubation: Overnight at 4°C

  • Washing: 3x5 minutes with PBS

  • Secondary antibody: Fluorophore-conjugated secondary antibody matching host species (typically anti-rabbit)

• Subcellular localization considerations:

  • MAB21L2 shows nuclear localization, particularly when BMP signaling is activated

  • Co-staining with SMAD1 can reveal interaction sites within the cell

• Tissue-specific recommendations:

  • Developmental tissue sections: MAB21L2 shows strong expression in developing eye, pharyngeal arches, and limb bud

  • Neural tissues: Important for neural tube formation experiments

  • P19 embryonic carcinoma cells: Good model system for studying MAB21L2 function

• Controls:

  • Positive control: Developmental tissues with known MAB21L2 expression

  • Negative control: Secondary antibody only

  • Counterstain: DAPI for nuclear visualization

This protocol integrates research findings on MAB21L2 localization with standard immunofluorescence methodologies.

What are the technical considerations for co-immunoprecipitation of MAB21L2 with its binding partners?

For successful co-immunoprecipitation (Co-IP) of MAB21L2 with its binding partners, particularly SMAD proteins, consider these technical aspects:

• Cell/tissue preparation:

  • Cell models: P19 cells show good MAB21L2-SMAD1 interaction

  • Treatment: BMP4 treatment enhances MAB21L2-SMAD1 interaction

  • Lysis buffer: Use buffer preserving protein-protein interactions (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, with protease/phosphatase inhibitors)

• Experimental design:

  • Forward IP: Use anti-MAB21L2 antibody for precipitation

  • Reverse IP: Use anti-SMAD1 antibody for precipitation

  • Detection: Western blot with antibody against the interaction partner

• Tag considerations:

  • Tagged constructs: Flag-tagged MAB21L2 and Myc-tagged SMAD1 have been successfully used

  • Detection strategy: Anti-flag for immunoprecipitation and anti-myc for immunodetection

• Protocol optimization:

  • Incubation time: 2-4 hours at 4°C for antibody binding

  • Washing stringency: Balance between removing non-specific binding and preserving interactions

  • Elution method: Consider non-denaturing elution if downstream applications require native protein

• Confirming specificity:

  • Input controls: 5-10% of pre-IP lysate

  • Negative controls: IgG from same species as the antibody

  • Positive controls: Known interactors (e.g., SMAD1-SMAD4 interaction)

This approach is based on published protocols that successfully demonstrated MAB21L2-SMAD1 interactions in both P19 cells and Xenopus embryos .

How can MAB21L2 antibodies be utilized to investigate BMP signaling pathway interactions?

MAB21L2 antibodies provide valuable tools for elucidating BMP signaling mechanisms through these research approaches:

• Pathway modulation studies:

  • Use MAB21L2 antibodies to monitor changes in MAB21L2-SMAD1 interaction following BMP4 stimulation or inhibition

  • Compare phosphorylated vs. total SMAD1 association with MAB21L2 to determine if SMAD activation affects interaction

• Chromatin immunoprecipitation (ChIP) applications:

  • Employ MAB21L2 antibodies for ChIP studies to identify genomic loci where MAB21L2 may repress transcription

  • Combine with SMAD1 ChIP for comparative occupancy analysis

• Temporal dynamics analysis:

  • Monitor MAB21L2-SMAD1 interaction timing following BMP stimulation

  • Track subcellular localization changes of MAB21L2 during BMP pathway activation

• Structure-function relationship investigation:

  • Map interaction domains using different MAB21L2 antibodies targeting specific epitopes

  • Compare wild-type vs. mutant MAB21L2 using antibodies that recognize conserved regions

• Comparative analysis across developmental contexts:

  Developmental Context	MAB21L2-SMAD1 Interaction	BMP Antagonism Effect
  Gastrulation	Enhanced with BMP4	Rescues dorsal axis
  Neural tube formation	Present	Not fully characterized
  Eye morphogenesis	Strong detection	Critical for normal development

This approach leverages MAB21L2's known role as a BMP4 antagonist and its interaction with SMAD1 to dissect signaling pathway dynamics in different developmental contexts .

What strategies can be employed to investigate MAB21L2's RNA-binding properties using antibodies?

To investigate MAB21L2's RNA-binding properties, implement these antibody-based experimental strategies:

• RNA immunoprecipitation (RIP):

  • Use MAB21L2 antibodies to precipitate protein-RNA complexes

  • Extract and analyze bound RNAs through sequencing or RT-PCR

  • Compare wild-type MAB21L2 to mutant forms that lose RNA-binding capability

• Subcellular localization studies:

  • Use immunofluorescence with MAB21L2 antibodies to track protein localization

  • Co-stain with RNA markers (e.g., poly(A) RNA FISH)

  • Analyze changes in localization following RNase treatment

• UV crosslinking and immunoprecipitation (CLIP):

  • UV-crosslink cells to stabilize protein-RNA interactions

  • Immunoprecipitate with MAB21L2 antibodies

  • Perform high-throughput sequencing of bound RNAs

• In vitro binding validation:

  • Purify MAB21L2 using affinity chromatography with antibodies

  • Perform binding assays with candidate RNA sequences

  • Compare binding properties of:

  MAB21L2 Form	Single-stranded RNA Binding	Functional Outcome
  Wild-type	Present	Normal development
  Mutant forms	Lost	Associated with developmental disorders

• RNA-protein complex visualization:

  • Use proximity ligation assay (PLA) with MAB21L2 antibodies and RNA probes

  • Perform immunofluorescence to visualize interaction sites within cells

These approaches capitalize on MAB21L2's demonstrated ability to bind single-stranded RNA while leveraging antibody specificity to isolate and characterize relevant complexes .

How can I design experiments to investigate the transcriptional repression activity of MAB21L2?

To explore MAB21L2's transcriptional repression function, implement these experimental designs:

• Reporter gene assays:

  • Use the established GAL4-DNA binding domain fusion system with MAB21L2

  • Compare with appropriate controls (GAL4 DBD alone, transcriptionally inactive fusion protein)

  • Measure luciferase activity reduction as indicator of repression

  • Quantitative data from published research:

  Construct	Relative Luciferase Activity	Cell Type
  GAL4 DBD alone	Baseline (100%)	COS7
  GAL4-MAB21L2	Significant reduction	COS7
  GAL4-MAB21L2	12-fold downregulation	P19

• Domain mapping experiments:

  • Create truncated or mutated versions of MAB21L2 fused to GAL4 DBD

  • Determine which regions are required for repression activity

  • Use MAB21L2 antibodies to confirm proper expression of fusion constructs

• Chromatin modification analysis:

  • Perform ChIP with antibodies against histone modifications at MAB21L2-targeted promoters

  • Compare active vs. repressive marks (H3K4me3 vs. H3K27me3)

  • Correlate with MAB21L2 binding using MAB21L2 antibodies

• Co-repressor identification:

  • Immunoprecipitate MAB21L2 with specific antibodies

  • Identify associated proteins by mass spectrometry

  • Validate interactions with co-IP and Western blotting

• Target gene identification:

  • Perform RNA-seq after MAB21L2 overexpression or knockdown

  • Combine with ChIP-seq using MAB21L2 antibodies

  • Integrate data to identify direct repression targets

This approach builds upon published evidence of MAB21L2's repressor activity when targeted to DNA via the GAL4 system, allowing for comprehensive characterization of its transcriptional regulation mechanisms .

What are the common challenges in MAB21L2 antibody applications and how can they be addressed?

Researchers frequently encounter these challenges when working with MAB21L2 antibodies, along with evidence-based solutions:

• Low signal intensity in Western blots:

  • Challenge: MAB21L2 detection can be difficult in certain tissues

  • Solutions:

    • Increase protein loading (30-40 μg)

    • Optimize antibody concentration (try higher end of recommended range)

    • Enhance signal with BMP4 treatment of cells/tissues

    • Use concentrated protein samples from tissues with known expression (e.g., mouse liver)

• Background or non-specific binding:

  • Challenge: Multiple bands or diffuse signal in immunoblotting

  • Solutions:

    • Increase blocking time and concentration (5-10% blocking agent)

    • Optimize primary antibody dilution (start with 1:2000)

    • Use more stringent washing (increase detergent concentration to 0.1% Tween-20)

    • Validate with peptide competition assays

• Inconsistent immunoprecipitation results:

  • Challenge: Variable MAB21L2-SMAD1 co-IP efficiency

  • Solutions:

    • Pre-clear lysates with protein G beads

    • Activate BMP signaling to enhance interaction

    • Optimize antibody-to-lysate ratio

    • Consider crosslinking to stabilize transient interactions

• Nuclear localization detection issues:

  • Challenge: Weak nuclear signal in immunofluorescence

  • Solutions:

    • Test different fixation methods (paraformaldehyde vs. methanol)

    • Optimize permeabilization (try 0.5% Triton X-100)

    • Use antigen retrieval techniques for tissue sections

    • Enhance with BMP pathway activation

• Antibody validation confidence:

  Validation Method	Recommended Approach	Expected Outcome
  Genetic knockout	CRISPR/siRNA MAB21L2 knockout cells	Loss of signal
  Overexpression	Transfected tagged MAB21L2	Enhanced signal
  Peptide competition	Pre-incubation with immunizing peptide	Reduced/eliminated signal
  Multi-antibody validation	Different antibodies targeting distinct epitopes	Concordant results

These troubleshooting approaches integrate published research methodologies with standard laboratory practices to enhance MAB21L2 antibody performance.

How can I validate the specificity of MAB21L2 antibodies for my experimental system?

To ensure MAB21L2 antibody specificity in your experimental system, implement this comprehensive validation strategy:

• Genetic manipulation controls:

  • Knockout/knockdown: Create MAB21L2-depleted samples using CRISPR-Cas9 or RNAi

  • Overexpression: Generate MAB21L2-overexpressing samples

  • Expected results: Signal should diminish with depletion and increase with overexpression

• Cross-reactivity assessment:

  • Western blot analysis across multiple species to confirm expected molecular weight (~40 kDa)

  • Testing in tissues with known expression patterns (developing eye, pharyngeal arches, limb bud)

  • Comparison with in situ hybridization data for correlation of protein and mRNA localization

• Epitope mapping validation:

  • For antibodies targeting specific regions (e.g., AA 1-300) :

    • Test against truncated protein constructs

    • Compare with antibodies targeting different epitopes

    • Validate detection of known MAB21L2 mutations

• Immunoprecipitation-mass spectrometry:

  • Immunoprecipitate with MAB21L2 antibody

  • Identify pulled-down proteins by mass spectrometry

  • Confirm presence of MAB21L2 and known interactors (e.g., SMAD1)

• Functional validation:

  • Block antibody access in functional assays (e.g., RNA binding assays)

  • Observe if MAB21L2-dependent functions are inhibited

  • Compare with results from genetic manipulation approaches

This multifaceted validation strategy ensures antibody specificity while leveraging known biological properties of MAB21L2, including its expression patterns, molecular interactions, and functional characteristics.

What controls should be included when studying MAB21L2-SMAD1 interactions?

When investigating MAB21L2-SMAD1 interactions, include these essential controls to ensure experimental validity:

• Expression controls:

  • Input lysate controls: Analyze 5-10% of pre-immunoprecipitation lysate to confirm protein expression

  • Western blot verification: Confirm MAB21L2 and SMAD1 expression levels before interaction studies

  • Expression normalization: Standardize protein levels across experimental conditions

• Immunoprecipitation controls:

  • Negative control antibody: Use isotype-matched IgG from the same species

  • Reverse co-IP: Perform reciprocal immunoprecipitation (pull down with anti-SMAD1, detect MAB21L2)

  • Known interaction controls: Include SMAD1-SMAD4 interaction as positive control

• Pathway modulation controls:

  • BMP4 treatment: Include both treated and untreated samples (interaction is enhanced by BMP4)

  • BMP inhibitor: Include BMP pathway inhibition to demonstrate specificity

  • Dose-response: Test multiple concentrations of pathway modulators

• Specificity controls:

  Control Type	Purpose	Expected Result
  SMAD4 direct binding	Test MAB21L2 specificity	No direct interaction
  Non-SMAD proteins	Test for non-specific binding	Minimal/no co-precipitation
  Competitive binding	Test if MAB21L2 competes with SMAD4	No competition observed

• Mutation/domain controls:

  • Truncated MAB21L2: Test which domains are required for interaction

  • SMAD1 phosphorylation mutants: Determine if receptor-mediated phosphorylation affects binding

  • Known MAB21L2 mutations: Test if disease-associated mutations alter interaction capability

These controls are based on published interaction studies between MAB21L2 and SMAD1, and will enhance the reliability and interpretability of your experimental findings .

How can MAB21L2 antibodies contribute to understanding eye development disorders?

MAB21L2 antibodies provide critical tools for investigating eye development disorders through these research applications:

• Developmental expression profiling:

  • Use immunohistochemistry with MAB21L2 antibodies to map spatiotemporal expression during eye development

  • MAB21L2 shows strong expression in the developing eye

  • Compare normal versus pathological developmental patterns

• Mutation impact assessment:

  • Generate antibodies recognizing specific MAB21L2 mutations associated with eye disorders

  • Compare protein localization and levels between wild-type and mutant forms

  • Assess whether mutations affect RNA-binding capability, which is lost in mutated forms

• Pathway disruption analysis:

  • Use MAB21L2 antibodies to monitor changes in SMAD1 interaction in disease models

  • Investigate BMP pathway alterations in ocular development disorders

  • Characterize molecular consequences of pathway disruption through co-localization studies

• Therapeutic intervention monitoring:

  • Employ MAB21L2 antibodies to track protein levels following experimental treatments

  • Monitor restoration of normal expression patterns after intervention

  • Use as biomarkers for treatment efficacy in developmental models

• Comparative analysis across eye development disorders:

  Disorder Type	MAB21L2 Expression Pattern	Associated Pathway Disruptions	Antibody Application
  Coloboma	Altered in specific regions	BMP signaling dysregulation	Regional expression analysis
  Microphthalmia	Potentially reduced levels	Growth signaling disruption	Quantitative immunoassays
  Developmental eye defects	Mislocalized protein	SMAD interaction alterations	Subcellular localization studies

These applications leverage MAB21L2's established role in eye morphogenesis and the availability of specific antibodies to advance understanding of developmental eye disorders .

What experimental approaches can elucidate the role of MAB21L2 in neural development?

To investigate MAB21L2's function in neural development, implement these antibody-based experimental strategies:

• Developmental timeline analysis:

  • Use immunohistochemistry with MAB21L2 antibodies to map expression throughout neural tube formation

  • Track protein levels during key developmental transitions

  • Correlate expression with neurulation milestones

• Neural differentiation studies:

  • Apply MAB21L2 antibodies in stem cell differentiation models

  • Monitor protein expression changes during neural lineage commitment

  • Use P19 embryonic carcinoma cells as established model system

• BMP pathway cross-regulation:

  • Investigate MAB21L2-SMAD1 interactions in neural contexts

  • Determine if MAB21L2's BMP4 antagonism affects neural patterning

  • Map expression relative to BMP gradient boundaries

• RNA-binding function analysis:

  • Identify neural-specific RNA targets using RIP with MAB21L2 antibodies

  • Compare wild-type versus mutant MAB21L2 RNA binding in neural cells

  • Investigate post-transcriptional regulation of neural development genes

• Experimental approach comparison:

  Approach	Technique	Expected Insights	Key Controls
  Loss-of-function	CRISPR/RNAi followed by IF	Requirements for neural development	Rescue experiments
  Gain-of-function	Overexpression with antibody detection	Sufficiency for neural induction	Pathway inhibitors
  Lineage tracing	MAB21L2 antibody combined with neural markers	Developmental trajectory	Multiple timepoints
  Mutant analysis	Antibodies detecting mutant vs. wild-type protein	Pathological mechanisms	Cross-validation

These approaches integrate MAB21L2's known developmental functions with neural-specific contexts to elucidate its role in neurulation and neural differentiation .

What emerging techniques might enhance MAB21L2 antibody applications in research?

Emerging technologies offer new possibilities for MAB21L2 research using antibodies:

• Single-cell protein analysis:

  • Single-cell Western blotting for heterogeneity analysis

  • Mass cytometry (CyTOF) with MAB21L2 antibodies for multiparameter phenotyping

  • Microfluidic immunoassays for quantifying MAB21L2 in rare cell populations

• Advanced imaging approaches:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging with cell-permeable MAB21L2 antibody fragments

  • Expansion microscopy for enhanced visualization of protein complexes

• Antibody engineering advances:

  • Nanobodies against MAB21L2 for improved penetration and reduced interference

  • Bispecific antibodies targeting MAB21L2 and interaction partners simultaneously

  • Recombinant antibody production for enhanced consistency

• In situ techniques:

  • Proximity ligation assay (PLA) for visualizing MAB21L2-SMAD1 interactions in tissue

  • Immuno-FISH combining MAB21L2 antibody staining with RNA detection

  • CODEX multiplexed protein imaging for developmental context

• Organoid applications:

  Organoid Type	MAB21L2 Investigation Focus	Antibody Application
  Retinal	Eye morphogenesis mechanisms	Developmental timeline studies
  Neural tube	BMP antagonism effects	Gradient analysis
  Cerebral	Cortical development role	Layer-specific expression

These emerging techniques will enable more precise, sensitive, and contextual analysis of MAB21L2 function across developmental processes and disease models.

How might integrative multi-omics approaches incorporating MAB21L2 antibodies advance developmental biology research?

Integrating MAB21L2 antibody-based studies with multi-omics approaches offers transformative research possibilities:

• Integrated genomics and proteomics:

  • Combine ChIP-seq using MAB21L2 antibodies with RNA-seq

  • Correlate genomic binding sites with transcriptional changes

  • Identify direct versus indirect regulatory targets

• Spatial transcriptomics integration:

  • Align MAB21L2 immunohistochemistry with spatial transcriptomics data

  • Map protein localization to gene expression domains

  • Create comprehensive developmental atlases with protein-RNA correlations

• Protein-RNA interactome mapping:

  • Integrate RIP-seq using MAB21L2 antibodies with CLIP-seq data

  • Correlate MAB21L2 binding with RNA fate

  • Construct regulatory networks incorporating transcriptional and post-transcriptional regulation

• Dynamic systems analysis:

  • Time-series experiments combining proteomics and transcriptomics

  • Model feedback loops involving MAB21L2 and BMP pathway components

  • Predict developmental transitions based on network states

• Multi-omics integration approaches:

  Data Integration	Technologies	Research Impact	Technical Considerations
  Protein-RNA-DNA	ChIP-seq, RIP-seq, ATAC-seq	Comprehensive regulatory landscape	Antibody specificity critical
  Temporal dynamics	Time-series proteomics and transcriptomics	Developmental transition mechanisms	Synchronization important
  Spatial context	Immunohistochemistry with spatial transcriptomics	Tissue-specific regulation patterns	Resolution matching required