PLAGL2 Antibody, HRP conjugated

What is PLAGL2 Antibody, HRP conjugated and what are its key specifications?

PLAGL2 Antibody, HRP conjugated is a polyclonal antibody raised in rabbits that specifically targets the amino acid region 324-469 of human PLAGL2 protein. The antibody is conjugated to horseradish peroxidase (HRP) enzyme, which facilitates detection in various immunoassays. Its specifications include:

• Target specificity: Human PLAGL2 (AA 324-469)

• Host species: Rabbit

• Clonality: Polyclonal

• Conjugation: HRP (Horseradish Peroxidase)

• Purity: >95%, Protein G purified

• Storage buffer: 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

• Storage conditions: -20°C or -80°C, avoid repeated freeze-thaw cycles

• Primary application: ELISA

How is the specificity of PLAGL2 Antibody determined and validated?

The specificity of PLAGL2 Antibody is determined through multiple validation steps:

• Immunogen specificity: The antibody is generated against a recombinant fragment of human PLAGL2 protein (amino acids 324-469), which defines its binding region.

• Purification methods: Protein G purification ensures that only IgG antibodies are retained, reducing non-specific binding.

• Cross-reactivity testing: The antibody is tested against human samples to confirm species reactivity.

• Application validation: The antibody is specifically validated for ELISA applications, ensuring reliable performance in this context.

• Control experiments: Proper validation includes positive and negative controls to confirm specific binding to PLAGL2 and absence of non-specific interactions.

For research applications, additional validation through Western blot, immunoprecipitation, or immunofluorescence might be necessary depending on the experimental context, though these are not the primary applications listed for this particular HRP-conjugated antibody .

What is the biological significance of PLAGL2 in cancer research?

PLAGL2 has emerged as a significant factor in cancer research for several reasons:

How can PLAGL2 Antibody, HRP conjugated be optimized for detecting nuclear versus cytosolic PLAGL2 expression?

Optimizing PLAGL2 Antibody, HRP conjugated for differential detection of nuclear versus cytosolic expression requires careful methodological considerations:

• Subcellular fractionation: Prior to ELISA, perform subcellular fractionation to separate nuclear and cytosolic components. This provides compartment-specific samples for analysis.

• Fixation protocols: When using this antibody in cell-based ELISA, optimize fixation protocols to preserve both nuclear and cytosolic epitopes. Paraformaldehyde (4%) typically preserves both compartments, while methanol enhances nuclear permeabilization.

• Permeabilization optimization: For cell-based ELISA, titrate detergent concentrations (Triton X-100 or saponin) to control membrane permeabilization without disrupting nuclear integrity.

• Quantitative comparison: Use standardized recombinant PLAGL2 proteins as calibrators to enable quantitative comparison between nuclear and cytosolic fractions.

• Validation with microscopy: Although this HRP-conjugated antibody is primarily for ELISA, parallel validation with immunofluorescence using unconjugated PLAGL2 antibodies can confirm localization patterns observed in ELISA results.

Research has shown that PLAGL2 localizes to both the nucleoplasm and cytosol in U251-MG glioblastoma cells, making this differential detection particularly relevant for understanding its function in different cellular compartments .

What are the considerations for using PLAGL2 Antibody in investigating the PLAGL2/MYCN/miR-506-3p regulatory axis?

When investigating the PLAGL2/MYCN/miR-506-3p regulatory axis, researchers should consider:

• Combined detection approaches: Utilize PLAGL2 Antibody in conjunction with MYCN detection methods to establish correlation between expression levels. This requires careful experimental design where both proteins are assessed in the same samples.

• Transfection studies: When manipulating miR-506-3p levels through transfection, assess PLAGL2 expression using the antibody to verify the miRNA regulatory effect.

• Sequential analysis: Design experiments to establish the sequence of regulatory events:

  • miR-506-3p directly targeting PLAGL2

  • PLAGL2 transcriptionally regulating MYCN

  • MYCN potentially regulating PLAGL2 in a feedback loop

• ChIP-PCR integration: While the HRP-conjugated antibody is designed for ELISA, parallel experiments using unconjugated PLAGL2 antibodies in ChIP-PCR can validate PLAGL2 binding to MYCN promoter regions.

• Controls for specificity: Include appropriate controls to distinguish direct from indirect regulatory effects in this complex axis.

• Retinoic acid treatment: Consider examining the effect of retinoic acid on this regulatory axis, as it has been shown to affect the expression of these components in neuroblastoma therapy contexts.

This regulatory axis is particularly relevant in neuroblastoma, where PLAGL2 has been identified as a direct target of miR-506-3p that mediates its regulation of MYCN expression .

How can PLAGL2 Antibody be used to assess the relationship between PLAGL2 expression and immune cell infiltration in cancer samples?

To investigate the relationship between PLAGL2 expression and immune cell infiltration using this antibody:

• Multiplex ELISA approaches: Develop multiplex ELISA protocols that simultaneously detect PLAGL2 and immune cell markers from the same sample.

• Sequential tissue sections: For tissue analysis, use sequential sections where one is analyzed for PLAGL2 using this HRP-conjugated antibody and adjacent sections for immune cell markers.

• Correlation with flow cytometry: Compare PLAGL2 expression levels determined by ELISA with flow cytometry data quantifying B cells, CD8+ T cells, CD4+ T cells, macrophages, DCs, and neutrophils from the same samples.

• Tissue microarray analysis: For larger sample sets, implement tissue microarray approaches to efficiently analyze PLAGL2 expression across multiple patient samples with varying immune infiltration profiles.

• Database integration: Correlate experimental PLAGL2 expression data with existing immune infiltration databases such as TIMER2.0 for comprehensive analysis.

• Functional validation: Design experiments to determine whether PLAGL2 expression directly influences immune cell recruitment or if the correlation is secondary to other factors.

Studies have shown correlations between PLAGL2 expression and the abundance of various immune cells, including B cells, CD8+ T cells, CD4+ T cells, macrophages, DCs, and neutrophils in high-grade glioma, suggesting potential immunomodulatory functions .

What are the critical steps for optimizing ELISA protocols using PLAGL2 Antibody, HRP conjugated?

Optimizing ELISA protocols with PLAGL2 Antibody, HRP conjugated requires attention to several critical steps:

• Antigen preparation: Ensure proper preparation of samples containing PLAGL2 protein:

  • For cell lysates: Use lysis buffers containing protease inhibitors to prevent degradation

  • For tissue samples: Optimize homogenization and extraction protocols

  • Consider subcellular fractionation if compartment-specific analysis is required

• Blocking optimization:

  • Test various blocking agents (BSA, non-fat milk, commercial blockers)

  • Determine optimal blocking time and temperature

  • Use sample diluent that contains blocking agent to minimize background

• Antibody titration:

  • Perform serial dilutions of the HRP-conjugated antibody to determine optimal concentration

  • Include positive controls with known PLAGL2 expression levels

  • Include negative controls lacking PLAGL2 to assess background signal

• Incubation conditions:

  • Determine optimal temperature (room temperature vs. 4°C)

  • Optimize incubation time (1-24 hours)

  • Consider whether agitation improves signal detection

• Washing steps:

  • Optimize wash buffer composition and pH

  • Determine optimal number and duration of washes

  • Ensure complete removal of wash buffer between steps

• Signal development and detection:

  • Select appropriate HRP substrate (TMB, ABTS, or others) based on sensitivity requirements

  • Optimize substrate incubation time

  • Determine optimal stopping point for colorimetric development

  • Calibrate plate reader settings for optimal signal detection

How should researchers address potential cross-reactivity concerns when using PLAGL2 Antibody in complex samples?

Addressing cross-reactivity concerns when using PLAGL2 Antibody requires a systematic approach:

• Pre-clearing samples:

  • Pre-adsorb samples with irrelevant proteins from the host species (rabbit) to reduce non-specific binding

  • Use species-specific blocking reagents when working with mixed-species samples

• Validation with genetic models:

  • Include PLAGL2 knockout or knockdown controls to confirm signal specificity

  • Test antibody reactivity in samples with known differential PLAGL2 expression

• Competitive binding assays:

  • Perform competition experiments using purified recombinant PLAGL2 protein

  • Decreasing signal with increasing competitor concentration confirms specificity

• Testing across multiple applications:

  • Compare results from different detection methods (ELISA, Western blot, immunofluorescence)

  • Consistent results across multiple techniques increase confidence in specificity

• Epitope mapping:

  • Understand that this antibody targets AA 324-469 of PLAGL2

  • Check sequence homology of this region with related proteins to predict potential cross-reactivity

  • Consider using additional antibodies targeting different PLAGL2 epitopes for confirmation

• Sequential immunodepletion:

  • Perform sequential immunodepletion with PLAGL2 and related proteins

  • Analyze the remaining signal to identify potential cross-reactive components

What methods can be used to quantitatively compare PLAGL2 expression across different cancer types using this antibody?

For quantitative comparison of PLAGL2 expression across cancer types using this HRP-conjugated antibody:

• Standard curve development:

  • Generate a standard curve using recombinant PLAGL2 protein (AA 324-469)

  • Ensure the curve covers the expected dynamic range of expression

  • Use appropriate curve-fitting models for accurate interpolation

• Sample normalization strategies:

  • Normalize to total protein concentration

  • Use housekeeping proteins as internal controls

  • Consider using tissue-specific normalization factors

• Batch control implementation:

  • Include common reference samples across all experimental batches

  • Use statistical methods to correct for batch effects

  • Process samples from different cancer types simultaneously when possible

• Multi-platform validation:

  • Correlate ELISA results with RT-qPCR data for PLAGL2 mRNA

  • Compare protein levels with publicly available transcriptomics data from databases like TIMER2.0, GENT2, ONCOMINE, and GEPIA

  • Validate with other quantitative protein methods like mass spectrometry

• Tissue microarray analysis:

  • Develop quantitative ELISA protocols compatible with tissue microarray formats

  • Enable high-throughput analysis across multiple cancer types

  • Incorporate spatial information about PLAGL2 expression

• Digital pathology integration:

  • Combine ELISA quantitation with digital pathology approaches

  • Correlate absolute protein levels with histological features

  • Develop algorithms for automated analysis across cancer types

Research has shown that PLAGL2 is upregulated in several cancers, including brain, breast, cervix, colon, esophagus, liver, lung, oral, ovary, and skin cancers, making such comparative studies particularly relevant .

How does PLAGL2 function as both an oncogene and tumor suppressor in different contexts?

PLAGL2's dual functionality as both an oncogene and tumor suppressor depends on cellular context and involves several mechanisms:

• Tissue-specific interactions:

  • In glioblastoma and colorectal cancer, PLAGL2 primarily functions as an oncogene through activation of the WNT/β-catenin pathway

  • In other contexts, its suppressive functions may emerge through distinct signaling networks

  • The antibody can help identify tissue-specific expression patterns correlating with these different functions

• Post-translational modifications:

  • Different modifications may alter PLAGL2 function

  • Phosphorylation, SUMOylation, or ubiquitination might switch its function between oncogenic and suppressive

  • While the HRP-conjugated antibody detects total PLAGL2, specialized antibodies for modified forms may be needed for complete functional characterization

• Protein-protein interactions:

  • PLAGL2 function may depend on available binding partners

  • Interaction with specific cofactors could determine whether it activates or represses target genes

  • Co-immunoprecipitation studies using non-conjugated PLAGL2 antibodies would be needed to identify these interactions

• Genomic context of binding sites:

  • PLAGL2 may activate or repress gene expression depending on the genomic context of its binding sites

  • The same transcription factor can have opposite effects at different promoters

  • ChIP-seq studies would complement antibody-based expression analysis to understand this context-dependence

• Concentration-dependent effects:

  • PLAGL2 may exhibit different functions at different expression levels

  • Quantitative analysis using the antibody could help establish thresholds for oncogenic versus suppressive functions

  • Dose-response studies in cellular models would be informative

Understanding this dual functionality is critical for developing PLAGL2-targeted therapies, as inhibition could be beneficial in some cancers but potentially harmful in others .

What is the significance of the amino acid region 324-469 targeted by this antibody in PLAGL2 function?

The amino acid region 324-469 targeted by this PLAGL2 antibody has particular significance:

• Structural implications:

  • This region lies outside the N-terminal C2H2 zinc finger domains responsible for DNA binding

  • It likely contains regulatory domains or protein interaction regions

  • Using this antibody enables specific detection of functions associated with this domain

• Post-translational modification sites:

  • This region may contain sites for phosphorylation, SUMOylation, or other modifications

  • Such modifications could regulate PLAGL2 activity or stability

  • The antibody might detect PLAGL2 regardless of modification status, depending on epitope accessibility

• Protein interaction domains:

  • AA 324-469 likely mediates interactions with cofactors or other regulatory proteins

  • These interactions could be critical for PLAGL2's transcriptional regulatory functions

  • Understanding these interactions provides insight into PLAGL2's role in different signaling pathways

• Species conservation:

  • Analysis of evolutionary conservation in this region provides insights into functional importance

  • Highly conserved regions suggest critical functional domains

  • The human specificity of this antibody reflects sequence differences in this region across species

• Alternative splicing considerations:

  • This region may be affected by alternative splicing events

  • The antibody might differentially detect PLAGL2 isoforms

  • Isoform-specific detection could reveal different functions in cellular processes

By targeting this specific region, researchers can examine the roles of these potential regulatory domains in PLAGL2's diverse cellular functions, providing a more nuanced understanding than antibodies targeting other regions of the protein .

How is PLAGL2 expression regulated at the transcriptional, post-transcriptional, and genomic levels?

PLAGL2 expression is regulated at multiple levels, providing several potential research avenues:

• Genomic regulation:

  • Copy number variation (CNV) at the PLAGL2 locus (20q11) contributes to its upregulation in cancers

  • Amplification of this chromosomal region is common in several malignancies

  • Genomic instability may lead to altered PLAGL2 expression

  • Comparing protein levels detected by the antibody with copy number data can reveal correlations

• Transcriptional regulation:

  • MYCN has been shown to transcriptionally regulate PLAGL2, suggesting a feedback loop

  • Other transcription factors likely control PLAGL2 expression in different cellular contexts

  • Promoter methylation status may influence transcription

  • ChIP studies can identify transcription factors binding to the PLAGL2 promoter

• Post-transcriptional regulation:

  • microRNAs, including miR-506-3p and miR-486-5p, directly regulate PLAGL2 mRNA

  • RNA-binding proteins (RBPs), such as upregulated human antigen R (HuR), affect PLAGL2 expression

  • The 3'-UTR of PLAGL2 plays a significant role in its expression regulation

  • Comparing PLAGL2 protein levels (via antibody detection) with mRNA levels can reveal post-transcriptional effects

• Post-translational regulation:

  • Protein stability factors influence PLAGL2 levels

  • Ubiquitination and proteasomal degradation pathways affect protein turnover

  • Functional analysis combining inhibitors of these pathways with antibody detection can elucidate these mechanisms

• Feedback mechanisms:

  • PLAGL2 may regulate its own expression through direct or indirect mechanisms

  • Complex regulatory networks involve PLAGL2, MYCN, and various miRNAs

  • Perturbation studies followed by antibody-based detection can map these networks

Understanding these multi-level regulatory mechanisms is essential for developing strategies to modulate PLAGL2 expression for therapeutic purposes, particularly in cancers where it functions as an oncogene .