vwc2l Antibody

What is VWC2L protein and what are its primary functions?

VWC2L (von Willebrand factor C domain containing 2 like) functions in the negative regulation of BMP signaling pathway and positive regulation of neuron differentiation. The protein is primarily localized in the extracellular space and forms part of the AMPA glutamate receptor complex. VWC2L is predominantly expressed in neural tissues including brain, central nervous system, hippocampus, hypothalamus, and thalamus . The protein plays a significant role in matrix mineralization, with differential expression patterns observed across various tissues, including higher expression in bone tissue (femur/calvaria) .

What types of VWC2L antibodies are currently available for research?

Multiple validated antibodies targeting VWC2L are commercially available, primarily as polyclonal antibodies produced in rabbit. These include antibodies from manufacturers like Invitrogen (catalog numbers PA5-63716, PA5-60838, PA5-120590), Atlas Antibodies (HPA044815), Novus Biologicals (NBP1-94166), St John's Laboratory (STJ119866), and antibodies-online (ABIN4365912) . Additionally, R&D Systems offers a sheep anti-human Brorin/VWC2 antibody (catalog number AF6147) that has been validated for Western blot and immunohistochemistry applications .

What are the most common applications for VWC2L antibodies?

VWC2L antibodies have been validated for several research applications including:

• Western blotting (WB): Most commonly used to detect VWC2L protein in tissue lysates, with human brain tissue showing a specific band at approximately 46kDa

• Immunohistochemistry (IHC): Used to localize VWC2L expression in fixed tissue sections, particularly in neuronal cells

• ELISA: Some antibodies have been validated for enzyme-linked immunosorbent assay applications

How should I optimize Western blot conditions when working with VWC2L antibodies?

When optimizing Western blot conditions for VWC2L detection:

• Sample preparation: Use tissue lysates from brain (ideally cerebellum or medulla) as positive controls, as VWC2L is highly expressed in these tissues

• Protein loading: Load approximately 25μg of protein per lane for optimal detection

• Antibody dilution: Start with a 1:500 to 1:2000 dilution range for primary antibody incubation

• Blocking conditions: Use 3% nonfat dry milk in TBST as an effective blocking buffer

• Detection system: Standard ECL detection systems are sufficient, with exposure times of approximately 60-90 seconds

• Expected band size: Look for a primary band at approximately 46kDa for human VWC2L, though the observed molecular weight may vary slightly (approximately 23kDa has been reported in some studies)

What controls should I include when performing immunohistochemistry with VWC2L antibodies?

For rigorous immunohistochemistry experiments with VWC2L antibodies:

• Positive tissue controls: Include brain tissue sections (particularly medulla or cerebellum) as positive controls

• Epitope retrieval method: Perform heat-induced epitope retrieval using basic antigen retrieval reagents before antibody incubation

• Antibody concentration: Start with 10 μg/mL concentration for overnight incubation at 4°C

• Detection system: Use an appropriate HRP-DAB staining kit, followed by hematoxylin counterstaining

• Negative controls: Include sections with omission of primary antibody and ideally tissues known not to express VWC2L

• Specificity validation: Look for specific staining localized to neurons in brain tissue samples

How can I validate VWC2L antibody specificity in my experimental system?

Multiple approaches should be implemented to validate VWC2L antibody specificity:

• Multi-antibody validation: Compare results using antibodies targeting different epitopes of VWC2L (there are multiple available antibodies targeting different regions)

• Molecular weight verification: Confirm band size in Western blot matches the expected molecular weight (approximately 23-46kDa, depending on isoform)

• Tissue expression pattern: Verify that expression patterns match known VWC2L distribution (high in brain, differential expression in bone tissues)

• Recombinant protein controls: If possible, use recombinant VWC2L protein as a positive control for antibody binding

• Transcript correlation: Compare protein expression with mRNA expression data from RT-PCR or RNA-seq experiments

• Knockdown validation: If feasible, demonstrate reduced antibody signal in cells/tissues with VWC2L knockdown

How can I distinguish between different VWC2L isoforms using antibodies?

Distinguishing between VWC2L isoforms requires careful experimental design:

• Isoform-specific antibodies: Select antibodies targeting unique regions present in specific isoforms. For example, exon 3 is present only in the Vwc2l-1 variant, so antibodies targeting this region would be specific to this isoform

• Molecular weight differentiation: The three known VWC2L transcript variants (Vwc2l-1, Vwc2l-2, and Vwc2l-3) may produce proteins of slightly different molecular weights, which can be resolved on higher-percentage polyacrylamide gels

• RT-PCR validation: Combine antibody-based detection with RT-PCR using isoform-specific primers to confirm expression patterns (e.g., forward primer: 5′-GGGATGGCTCTTCATATTCATGAAGC-3′, reverse primer: 5′-CACAGTCTGCTTGCCTTGGCATTCGC-3′)

• Tissue-specific expression: Leverage the known differential expression patterns of isoforms across tissues (e.g., Vwc2l-2 is more highly expressed in femur/calvaria compared to Vwc2l-1, while Vwc2l-3 is more highly expressed in lung and heart)

What are the best approaches for studying VWC2L's role in matrix mineralization?

To investigate VWC2L's role in matrix mineralization:

• Overexpression studies: Transfect osteoblastic cell lines (such as MC3T3-E1) with VWC2L expression vectors to observe effects on mineralization timing and extent

• Time-course experiments: Monitor VWC2L expression changes during the differentiation and mineralization process using real-time PCR and protein detection methods

• Mineralization assay: Use Alizarin Red S staining to visualize mineralized nodule formation at various time points (e.g., days 14, 21, 28) following VWC2L manipulation

• Isoform-specific effects: Compare the effects of different VWC2L isoforms on mineralization by using isoform-specific constructs

• Protein purification: For mechanistic studies, purify VWC2L protein using nickel column purification followed by size exclusion chromatography for functional assays

• Expression correlation: Quantify the relationship between VWC2L expression levels and mineralization extent using image analysis of mineralized nodules

How can I investigate VWC2L's interactions with the BMP signaling pathway?

For studying VWC2L's role in BMP signaling inhibition:

• Signaling reporter assays: Use BMP-responsive luciferase reporters to quantify the inhibitory effect of VWC2L on BMP signaling

• Co-immunoprecipitation: Investigate physical interactions between VWC2L and BMP pathway components using antibodies for immunoprecipitation followed by Western blot analysis

• Dose-dependent studies: Apply varying concentrations of purified VWC2L protein to cells and measure changes in downstream BMP signaling markers

• Pathway component analysis: Assess changes in phosphorylation states of SMAD proteins (key BMP signal transducers) in the presence of VWC2L

• Domain mutation studies: Create VWC2L constructs with mutations in specific domains to identify regions essential for BMP inhibition

• Competitive binding assays: Determine if VWC2L competes with BMP receptors for BMP ligand binding

Why might I observe multiple bands when performing Western blot for VWC2L?

Multiple bands in VWC2L Western blots may occur for several reasons:

• Isoform detection: VWC2L exists in at least three transcript variants (Vwc2l-1, Vwc2l-2, and Vwc2l-3) with differential tissue expression, potentially resulting in multiple specific bands

• Post-translational modifications: VWC2L may undergo glycosylation or other modifications that alter its migration pattern

• Proteolytic processing: As an extracellular protein, VWC2L may undergo proteolytic cleavage resulting in fragments of different sizes

• Non-specific binding: Some antibodies may cross-react with similar proteins, particularly other von Willebrand factor domain-containing proteins

• Sample preparation issues: Incomplete denaturation or sample degradation can result in aberrant banding patterns

To address these issues, validate with multiple antibodies, include appropriate positive controls (brain tissue lysates), optimize blocking conditions, and consider using freshly prepared samples under reducing conditions .

What are the common pitfalls when detecting VWC2L in immunohistochemistry applications?

Common challenges in VWC2L immunohistochemistry include:

• Insufficient epitope retrieval: VWC2L detection often requires heat-induced epitope retrieval using basic antigen retrieval reagents

• Background staining: Optimize blocking conditions and antibody concentrations (start with 10 μg/mL) to minimize non-specific binding

• Weak signal: VWC2L is primarily localized to neurons in brain tissue; ensure sample preparation preserves protein antigenicity

• Fixation artifacts: Overfixation may mask epitopes; consider testing multiple fixation protocols

• Antibody specificity: Validate antibody using Western blot before immunohistochemistry applications to confirm target specificity

• Detection system sensitivity: Use amplification systems like HRP-DAB for optimal visualization of lower-abundance proteins

How can I optimize RT-PCR conditions for detecting different VWC2L transcript variants?

For optimal RT-PCR detection of VWC2L transcript variants:

• Primer design: Use primers that can distinguish between variants (e.g., for Vwc2l-1 specific detection, target exon 3)

  • For general VWC2L detection: forward primer: 5′-GGGATGGCTCTTCATATTCATGAAGC-3′, reverse primer: 5′-CACAGTCTGCTTGCCTTGGCATTCGC-3′

  • For transcript variant cloning: forward primer, 5′-GCATTCCCATGGAAGGAGCTGAGTGTG-3′ and reverse primer, 5′-CAACCCCAGAAGGTTTTGCTGGTGACC-3′

• PCR conditions:

  • Initial denaturation: 15 min at 95°C

  • Cycling conditions: 30 s at 95°C, 1 min at 60°C, 1.5 min at 72°C for 30 cycles

• Template selection: Use tissue-specific cDNA based on known expression patterns (brain for Vwc2l-1, femur/calvaria for Vwc2l-2, lung/heart for Vwc2l-3)

• Quantitative analysis: For precise quantification, use real-time PCR with specific primers-probe systems (e.g., Mm01260094_m1 for Vwc2l-1) and appropriate reference genes like GAPDH

• Validation: Confirm PCR products by sequencing to ensure specificity, particularly when studying novel tissue sources

How does VWC2L expression correlate with tissue development and pathological conditions?

Analysis of VWC2L expression patterns reveals:

• Tissue-specific distribution: VWC2L isoforms show distinct tissue expression patterns:

  • Vwc2l-1: Highest expression in brain tissues

  • Vwc2l-2: Higher expression in skeletal tissues (femur/calvaria)

  • Vwc2l-3: Higher expression in lung and heart tissues

• Developmental regulation: In osteoblastic cell differentiation, Vwc2l-1 expression increases markedly during late mineralization stages (~7-fold at day 28 and ~18-fold at day 35 compared to day 7)

• Functional correlation: Increased VWC2L expression correlates with accelerated mineralized nodule formation, suggesting a potential role in osteoblast differentiation and bone development

• Neuronal expression: In brain tissues, VWC2L expression is specifically localized to neurons, indicating potential roles in neuronal function or development

When analyzing VWC2L expression data, consider tissue context, developmental stage, and which isoform is being detected, as these factors significantly impact interpretation.

What are the key considerations when comparing VWC2L detection across different antibodies?

When comparing results obtained with different VWC2L antibodies:

• Epitope differences: Antibodies targeting different regions of VWC2L may yield different results based on epitope accessibility in various experimental conditions

• Comparative validation table:

  Antibody Source	Catalog Number	Host Species	Validated Applications	Target Epitope Region
  Invitrogen	PA5-120590	Rabbit	WB	Human, Mouse
  Invitrogen	PA5-63716	Rabbit	IHC	Human
  Atlas Antibodies	HPA044815	Rabbit	IHC	Human
  R&D Systems	AF6147	Sheep	WB, IHC	Human (Ser28-Met325)
  ABclonal	A17853	Rabbit	WB, ELISA	Mouse (aa 1-50)

• Performance variation: Differences in sensitivity, specificity, and background may occur between antibodies, requiring optimization for each application

• Isoform detection: Some antibodies may preferentially detect certain VWC2L isoforms based on their epitope location, potentially leading to divergent results

• Cross-reactivity: Consider potential cross-reactivity with related proteins, particularly other von Willebrand factor domain-containing proteins

• Validation standards: Prioritize antibodies with multiple validation methods (Western blot, IHC, knockout controls) when selecting reagents for critical experiments

How can I integrate VWC2L protein expression data with functional studies?

To effectively integrate VWC2L expression and functional data:

• Correlation analysis: Quantitatively correlate VWC2L expression levels with functional outcomes:

  • For mineralization studies: Measure Alizarin Red S staining intensity and correlate with VWC2L expression levels

  • For neuronal studies: Correlate VWC2L expression with measures of neuronal differentiation or function

• Temporal relationships: Establish temporal relationships between VWC2L expression changes and functional outcomes:

  • In osteoblast differentiation, increased VWC2L expression precedes or coincides with mineralization

  • Document time-course relationships through parallel sampling for protein expression and functional assays

• Cause-effect validation: Confirm causality through gain and loss of function approaches:

  • Overexpression: VWC2L overexpression accelerates mineralized nodule formation in osteoblastic cells

  • Knockdown/knockout: Design experiments to assess phenotypes upon VWC2L reduction

• Mechanistic connections: Link VWC2L to known pathways:

  • BMP signaling: Measure BMP pathway activity markers in relation to VWC2L expression

  • AMPA receptor complex: Investigate glutamatergic signaling in relation to VWC2L levels

• Isoform-specific functions: Distinguish functions of different VWC2L isoforms by correlating isoform-specific expression with distinct functional outcomes