PLAGL2 Antibody, Biotin conjugated

What is PLAGL2 and why is it significant in research?

PLAGL2 (Pleiomorphic adenoma-like protein 2) is a zinc finger protein that shows weak transcriptional activatory activity . This protein, also known by its alias KIAA0198, plays significant roles in epigenetic regulation and nuclear signaling pathways . Research interest in PLAGL2 has grown due to its implications in cellular processes including differentiation, proliferation, and apoptosis. The biotin-conjugated antibody targeting PLAGL2 allows researchers to detect and study this protein in various experimental contexts, particularly useful for investigating transcriptional regulation mechanisms.

What are the structural and functional characteristics of the PLAGL2 Antibody, Biotin conjugated?

The PLAGL2 Antibody (Biotin conjugated) is a polyclonal antibody raised in rabbits against recombinant Human Zinc finger protein PLAGL2 protein (specifically amino acids 324-469) . The antibody has an IgG isotype and is conjugated with biotin, which enhances detection sensitivity through avidin-biotin complexing techniques . The antibody specifically targets human PLAGL2 and is purified using Protein G affinity chromatography to >95% purity . Its functional activity allows for detection of PLAGL2 in experimental settings, primarily validated for ELISA applications . The biotin conjugation enables versatile detection methods including streptavidin-based visualization systems.

How does the biotin conjugation affect the antibody's functionality compared to other conjugates?

Biotin conjugation provides several methodological advantages over other conjugates such as HRP (Horseradish Peroxidase):

The biotin-conjugated antibody offers superior signal amplification through the strong biotin-streptavidin interaction (Kd ≈ 10^-15 M), making it particularly valuable for detecting low-abundance proteins like transcription factors . This conjugation method also provides more flexibility in experimental design than direct enzyme conjugates.

What are the validated experimental applications for PLAGL2 Antibody, Biotin conjugated?

While the PLAGL2 Antibody, Biotin conjugated has been primarily validated for ELISA applications , researchers can potentially adapt it for other techniques with proper validation:

When adapting this antibody for non-validated applications, thorough controls and optimization steps are essential. For instance, when attempting immunohistochemistry, researchers should test multiple antigen retrieval methods and antibody dilutions to determine optimal signal-to-noise ratios.

What is the recommended methodology for ELISA using PLAGL2 Antibody, Biotin conjugated?

For optimal ELISA results with PLAGL2 Antibody, Biotin conjugated, follow this methodological approach:

• Plate Coating: Coat microplate wells with target antigen (recombinant PLAGL2 or sample containing PLAGL2) in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 3-5% BSA in PBST for 1-2 hours at room temperature.

• Primary Antibody: Apply diluted PLAGL2 Antibody, Biotin conjugated in blocking buffer. Start with 1:1000 dilution and optimize as needed. Incubate for 2 hours at room temperature.

• Detection System: Use streptavidin-HRP (typically 1:5000-1:10000 dilution) for 1 hour at room temperature.

• Substrate Development: Add TMB substrate and monitor color development.

• Signal Reading: After stopping the reaction with 2N H₂SO₄, read absorbance at 450nm.

Critical optimization parameters include antibody dilution, incubation time, and washing stringency. The buffer composition (0.01M PBS, pH 7.4 with 0.03% Proclin 300 preservative) should be considered when designing experiments to avoid buffer incompatibilities .

How should researchers properly store and handle the PLAGL2 Antibody, Biotin conjugated to maintain its activity?

Proper storage and handling of PLAGL2 Antibody, Biotin conjugated is crucial for maintaining its activity:

• Long-term Storage: Store at -20°C or -80°C in the provided buffer (50% Glycerol, 0.01M PBS, pH 7.4) .

• Working Aliquots: Upon receipt, divide into small working aliquots before freezing to avoid repeated freeze-thaw cycles.

• Thawing Procedure: Thaw aliquots rapidly at room temperature and place on ice immediately after thawing.

• Working Dilution Stability: Diluted antibody should be used within 24 hours and not stored for future use.

• Temperature Sensitivity: Avoid elevated temperatures during handling as biotin conjugates can be sensitive to thermal degradation.

Critical note: The manufacturer explicitly warns against repeated freeze-thaw cycles , as each cycle can reduce antibody activity by approximately 10-15%, potentially compromising experimental results.

How can researchers validate PLAGL2 Antibody specificity in their experimental systems?

Validation of antibody specificity is crucial for meaningful experimental outcomes. For PLAGL2 Antibody, Biotin conjugated, implement these validation strategies:

• Positive Controls: Use cell lines with known PLAGL2 expression (e.g., HeLa cells) alongside experimental samples.

• Negative Controls: Include samples from PLAGL2 knockout systems or cells with siRNA-mediated PLAGL2 knockdown.

• Peptide Competition Assay: Pre-incubate antibody with excess immunizing peptide (amino acids 324-469 of human PLAGL2) to confirm binding specificity.

• Cross-reactivity Assessment: Test antibody against closely related proteins, particularly PLAG1 and PLAGL1, which share structural similarities with PLAGL2.

• Molecular Weight Verification: Confirm detection at the expected molecular weight (approximately 60-65 kDa for human PLAGL2).

Remember that this antibody was raised against human PLAGL2 (324-469AA) , so validation is particularly important when studying non-human samples or when examining full-length protein interactions.

What are the considerations for studying PLAGL2 in the context of epigenetic and nuclear signaling research?

PLAGL2 functions within complex epigenetic and nuclear signaling networks , requiring specialized experimental design:

• Chromatin Immunoprecipitation (ChIP) Adaptations:

  • For biotin-conjugated antibodies in ChIP, use streptavidin-based magnetic beads

  • Implement stringent blocking with free biotin in nuclear lysates to prevent endogenous biotin interference

  • Consider sequential ChIP to identify PLAGL2 co-regulators

• Transcriptional Activity Assessment:

  • Combine PLAGL2 detection with RNA polymerase II co-localization studies

  • Implement genome-wide approaches like ChIP-seq with biotin-based pull-down systems

  • Correlate PLAGL2 binding with histone modification patterns (H3K4me3, H3K27ac)

• Protein-Protein Interaction Studies:

  • Use proximity ligation assays with the biotin-conjugated antibody

  • Implement RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) with streptavidin pull-down

  • Consider BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in living cells

The weak transcriptional activatory activity of PLAGL2 suggests it may function within larger multiprotein complexes, necessitating careful experimental design to capture these interactions.

What methodological approaches can be used to study PLAGL2 post-translational modifications?

Studying PLAGL2 post-translational modifications (PTMs) requires specialized protocols when using biotin-conjugated antibodies:

• PTM-specific Detection Strategy:

  • Two-step detection: First use PLAGL2 Antibody, Biotin conjugated to capture total PLAGL2

  • Then probe with modification-specific antibodies (phospho, acetylation, SUMOylation)

• Mass Spectrometry Workflow:

  • Streptavidin pull-down of biotin-labeled PLAGL2

  • On-bead digestion to preserve labile modifications

  • Titanium dioxide enrichment for phosphopeptides

  • Analysis by LC-MS/MS with electron transfer dissociation for precise modification mapping

• Functional Impact Assessment:

  • Correlation of PTM status with PLAGL2 localization (nuclear vs. cytoplasmic)

  • Examination of PTM changes during cell cycle progression

  • Analysis of modification-dependent protein-protein interactions

This methodological approach addresses the significant technical challenge of simultaneously detecting a specific protein and its modifications when working with conjugated primary antibodies.

What are common technical issues with biotin-conjugated antibodies in PLAGL2 detection and how can they be resolved?

Researchers frequently encounter these technical issues when working with PLAGL2 Antibody, Biotin conjugated:

When troubleshooting, implement systematic changes to one variable at a time while maintaining appropriate controls. Document all optimization steps carefully for reproducibility and method reporting.

How can researchers optimize protocols when transitioning from ELISA to other applications with PLAGL2 Antibody, Biotin conjugated?

When adapting the PLAGL2 Antibody, Biotin conjugated from its validated ELISA application to other techniques, follow these methodological transitions:

• From ELISA to Western Blotting:

  • Increase antibody concentration 2-3 fold from ELISA working dilution

  • Extend primary antibody incubation to overnight at 4°C

  • Use milk-free blocking buffer (BSA-based) to prevent biotin-milk protein interactions

  • Implement streptavidin-HRP detection with enhanced chemiluminescence

• From ELISA to Immunofluorescence:

  • Optimize fixation method (4% paraformaldehyde typically works best)

  • Test multiple antigen retrieval methods (citrate, EDTA, Tris)

  • Use fluorophore-conjugated streptavidin (Alexa Fluor 488/555/647)

  • Include Sudan Black B treatment to reduce autofluorescence

• From ELISA to Flow Cytometry:

  • Increase antibody concentration 5-10 fold from ELISA working dilution

  • Extend incubation time (45-60 minutes)

  • Use saponin-based permeabilization for intracellular PLAGL2 detection

  • Implement multi-parameter analysis to correlate PLAGL2 with other markers

Document all optimization steps methodically, as the transition process itself generates valuable protocol information for the research community.

What advanced control experiments should be included when studying PLAGL2 in transcriptional regulation contexts?

When using PLAGL2 Antibody, Biotin conjugated for transcriptional regulation studies, implement these advanced control experiments:

• Functional Validation Controls:

  • PLAGL2 overexpression vs. knockdown/knockout gene expression profiles

  • Rescue experiments with wild-type vs. mutant PLAGL2

  • Correlation of PLAGL2 binding with actual transcriptional output

• Interaction Specificity Controls:

  • Sequential ChIP with other transcription factors

  • Competitive binding assays with known PLAGL2 binding partners

  • DNA-protein interaction validation using EMSA with supershift

• Context-dependent Regulation Controls:

  • Cell cycle synchronization to assess phase-specific functions

  • Stimulation/inhibition of relevant signaling pathways

  • Comparison across multiple cell types with varying PLAGL2 expression levels

These controls address the challenge of distinguishing direct PLAGL2 effects from indirect consequences of experimental manipulation, particularly important given its weak transcriptional activatory properties .

How might PLAGL2 Antibody, Biotin conjugated be utilized in emerging epigenetic research methodologies?

The PLAGL2 Antibody, Biotin conjugated has significant potential in emerging methodologies:

• Spatial Transcriptomics Applications:

  • Integration with Visium or Slide-seq platforms for spatial mapping of PLAGL2 activity

  • Correlation of PLAGL2 localization with zone-specific gene expression patterns

  • Development of multiplex imaging with other epigenetic regulators

• Single-Cell Applications:

  • Adaptation for CyTOF (mass cytometry) using metal-tagged streptavidin

  • Implementation in single-cell Western blotting platforms

  • Development of PLAGL2 activity sensors for live-cell imaging

• Multi-omics Integration:

  • Correlation of PLAGL2 binding sites (ChIP-seq) with chromatin accessibility (ATAC-seq)

  • Integration with RNA-seq to create comprehensive regulatory networks

  • Combination with proteomics to map PLAGL2-dependent protein expression changes

These emerging methodologies would benefit from the signal amplification properties of biotin-conjugated antibodies, particularly for detecting low-abundance transcription factors like PLAGL2 in spatially resolved or single-cell contexts .

What are methodological considerations for studying PLAGL2 across different model systems?

Studying PLAGL2 across diverse model systems requires careful methodological adaptation:

Since the antibody was developed against human PLAGL2 (324-469AA) , cross-species applications require rigorous validation. When working with non-human systems, researchers should first perform Western blot analysis to confirm appropriate recognition of the target protein at the expected molecular weight before proceeding to more complex applications.

How can computational approaches enhance research utilizing PLAGL2 Antibody, Biotin conjugated?

Computational approaches can significantly enhance PLAGL2 research:

• Prediction-Validation Pipeline:

  • Use computational prediction of PLAGL2 binding motifs

  • Validate predictions experimentally using PLAGL2 Antibody, Biotin conjugated

  • Refine algorithms based on experimental results

• Network Analysis Integration:

  • Map PLAGL2 to broader epigenetic regulation networks

  • Identify potential co-factors through interactome analysis

  • Predict functional outcomes of PLAGL2 binding

• Image Analysis Optimization:

  • Develop machine learning algorithms for automated PLAGL2 localization

  • Implement nuclear segmentation for quantitative analysis

  • Create computational workflows for co-localization studies

• Multi-omics Data Integration:

  • Correlate PLAGL2 binding patterns with histone modifications

  • Integrate with transcriptomic data to build regulatory models

  • Develop predictive models of PLAGL2-dependent phenotypes