PRDM10 Antibody, HRP conjugated

What is PRDM10 and why is it important in developmental biology?

PRDM10 is a PR domain-containing zinc finger protein that functions as a sequence-specific transcription factor. It plays a critical role during early embryonic development, particularly in mouse embryonic stem cells (mESCs). Research has established that PRDM10 regulates global translation through direct transcriptional control of translation initiation factors such as Eif3b. This regulation is essential for mESC survival and early embryonic development . The protein contains a characteristic PR/SET domain along with multiple zinc finger motifs that enable DNA binding specificity. Understanding PRDM10 function provides insights into fundamental developmental processes and stem cell biology.

What are the key specifications of commercially available PRDM10 HRP-conjugated antibodies?

PRDM10 HRP-conjugated antibodies are specialized immunological reagents designed for enhanced detection sensitivity without requiring secondary antibodies. Typical specifications include:

These specifications ensure reliable detection of PRDM10 in experimental settings, particularly for quantitative analyses requiring high sensitivity.

What experimental applications are most suitable for PRDM10 HRP-conjugated antibodies?

PRDM10 HRP-conjugated antibodies are primarily optimized for ELISA applications, where direct detection eliminates additional incubation steps required with unconjugated primary antibodies . The HRP conjugation enables direct enzymatic color development when appropriate substrates (such as TMB or DAB) are added. While ELISA represents the validated application, researchers should note that:

• The HRP conjugation makes these antibodies potentially suitable for direct immunohistochemistry (IHC) applications without secondary antibodies.

• Western blotting may be possible with direct detection protocols, though signal amplification will be more limited than two-step detection systems.

• Chromatin immunoprecipitation (ChIP) applications may be challenging with HRP-conjugated antibodies, where unconjugated variants are typically preferred.

For applications beyond ELISA, preliminary validation experiments are strongly recommended before proceeding with full experimental protocols.

How can PRDM10 antibodies be employed to study its gene regulatory functions?

PRDM10 functions as a sequence-specific transcription factor that binds to promoter regions and regulates gene expression. To study these regulatory functions:

• Chromatin Immunoprecipitation (ChIP): Unconjugated PRDM10 antibodies can be used for ChIP-seq analysis to identify genome-wide binding sites. Research has shown that approximately 70.8% of PRDM10 binding sites reside within 1 kb upstream or overlapping with gene transcriptional start sites (TSSs) . This approach has revealed that PRDM10 binding is associated with regions of transcriptionally active chromatin marked by H3K4me3, H3K4me1, and H3K36me3 .

• Promoter Activity Assays: Following identification of PRDM10 binding sites, reporter assays can be used to measure transcriptional activation. Studies have demonstrated that PRDM10 can strongly activate reporter constructs containing its consensus motif, with mutation of this sequence abolishing PRDM10-dependent activation .

• Protein-DNA Interaction Analysis: Gel shift assays using recombinant PRDM10 protein fragments (particularly those containing the central zinc finger array) have confirmed direct binding to its cognate DNA motif . This binding can be specifically competed with excess unlabeled wild-type probe but not with mutated sequences.

These techniques collectively enable comprehensive characterization of PRDM10's gene regulatory networks and molecular mechanisms of action.

What methodological considerations are important when performing ELISA with PRDM10 HRP-conjugated antibodies?

When performing ELISA with PRDM10 HRP-conjugated antibodies, several critical methodological considerations should be addressed:

• Optimization of Antibody Concentration: Titration experiments should establish the optimal concentration for maximum signal-to-noise ratio. Starting with 1:1000 dilution is recommended, with further optimization between 1:500-1:5000 .

• Sample Preparation:

  • Cell lysates should be prepared using buffers containing protease inhibitors to prevent PRDM10 degradation

  • For tissue samples, standardized extraction protocols with proper homogenization are essential

  • Recombinant protein standards should be included for quantitative analysis

• Blocking Considerations: Since PRDM10 antibodies may exhibit different background characteristics than unconjugated antibodies, optimization of blocking solutions is critical:

  • 3-5% BSA in PBST is generally recommended

  • Milk-based blockers may interfere with some HRP detection systems

  • Control wells without primary antibody help establish background levels

• Detection Optimization:

  • Substrate selection affects sensitivity (TMB offers highest sensitivity)

  • Incubation time requires careful monitoring to prevent signal saturation

  • Immediate reading after stop solution addition prevents signal deterioration

• Data Analysis:

  • Four-parameter logistic regression models typically provide best fit for standard curves

  • Baseline correction against negative controls improves quantification accuracy

These considerations ensure reliable and reproducible ELISA results when working with PRDM10 HRP-conjugated antibodies.

How can researchers validate the specificity of PRDM10 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For PRDM10 antibodies, a comprehensive validation strategy should include:

• Genetic Validation: The gold standard approach involves comparing antibody signal in wild-type versus PRDM10-depleted or knockout cells. ChIP-seq experiments have demonstrated that PRDM10 binding peaks detected in wild-type cells are absent or strongly diminished in PRDM10-depleted cells .

• Peptide Competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals. For C-terminal PRDM10 antibodies, peptides with sequence C-NGNGSSEVHITKP can be used as competitors .

• Multiple Antibody Validation: Using antibodies targeting different epitopes of PRDM10 should produce consistent results. Research has utilized antibodies recognizing N-terminal (AA 1-122) and C-terminal regions with compatible findings .

• Western Blot Analysis: Detection of a single band at the expected molecular weight (~130 kDa) provides verification of specificity . Multiple bands may indicate degradation products or cross-reactivity.

• Cross-Species Reactivity Assessment: Testing the antibody across human, mouse, and rat samples helps confirm conservation of the recognized epitope and broadens applicability. Available PRDM10 antibodies have demonstrated varied cross-reactivity profiles .

Implementing these validation approaches ensures that experimental observations truly reflect PRDM10 biology rather than antibody artifacts.

What experimental protocols maximize successful chromatin immunoprecipitation (ChIP) with PRDM10 antibodies?

While HRP-conjugated antibodies are not typically used for ChIP applications, unconjugated PRDM10 antibodies have been successfully employed in ChIP-seq experiments. Based on published protocols , the following optimizations are recommended:

• Crosslinking Optimization:

  • Use 1% formaldehyde for 10 minutes at room temperature for most applications

  • For detection of weaker or transient interactions, consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Chromatin Fragmentation:

  • Sonication should aim for fragments between 200-500 bp

  • Optimization of sonication cycles is critical (typically 15-20 cycles of 30 seconds on/30 seconds off)

  • Verify fragmentation by agarose gel electrophoresis before proceeding

• Antibody Selection and Validation:

  • Validate multiple polyclonal antibodies against different PRDM10 epitopes

  • For ChIP-seq, antibodies recognizing the N-terminal region have shown success

  • Include IgG negative controls and positive controls (such as antibodies against histone marks)

• IP Conditions:

  • Optimize antibody concentration (typically 3-5 μg per IP reaction)

  • Extended incubation (overnight at 4°C) improves recovery

  • Include protease inhibitors and phosphatase inhibitors in all buffers

• Washing Stringency:

  • Progressive washing with increasing salt concentrations removes non-specific interactions

  • Final high-stringency washes (LiCl buffer) are essential for specificity

These optimizations have enabled identification of 528 reproducible PRDM10 binding peaks in mouse embryonic stem cells, with strong enrichment at promoter regions .

What approaches can resolve contradictory results when using different PRDM10 antibodies?

Researchers occasionally encounter contradictory results when using different PRDM10 antibodies. A systematic troubleshooting approach includes:

• Epitope Mapping Analysis:

  • Different antibodies recognize distinct epitopes that may be differentially accessible

  • N-terminal antibodies (AA 1-122) versus C-terminal antibodies (NGNGSSEVHITKP) may yield different results based on protein conformation or interactions

  • Document which protein domains each antibody targets and correlate with functional domains

• Isoform-Specific Detection:

  • PRDM10 exists in multiple isoforms (NP_064613.2, NP_955469.1, NP_955470.1, NP_955471.1)

  • Some antibodies may recognize all isoforms while others are isoform-specific

  • Correlate antibody recognition sites with isoform-specific sequences

• Experimental Condition Optimization:

  • Fixation conditions affect epitope accessibility (critical for IHC/ICC)

  • Denaturing conditions in Western blotting may destroy conformational epitopes

  • Native versus reducing conditions significantly impact detection sensitivity

• Cross-Validation Approaches:

  • Implement orthogonal detection methods (mass spectrometry, RNA-seq)

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to validate specificity

  • Compare antibody performance across multiple experimental platforms

• Statistical Analysis of Reproducibility:

  • Apply IDR (Irreproducible Discovery Rate) analysis to ChIP-seq data from different antibodies

  • Establish confidence thresholds (e.g., IDR < 0.05) for peak calling

  • Focus on consensus results detected by multiple antibodies

This systematic approach helps distinguish genuine biological findings from antibody-specific artifacts.

How can researchers optimize detection sensitivity when using PRDM10 HRP-conjugated antibodies?

Maximizing detection sensitivity with PRDM10 HRP-conjugated antibodies requires careful optimization at multiple experimental stages:

• Signal Amplification Systems:

  • Tyramide Signal Amplification (TSA) can increase sensitivity 10-100 fold

  • Enhanced chemiluminescent substrates (SuperSignal West Femto) provide greater sensitivity than standard ECL

  • Polymer-based detection systems offer improved signal without increased background

• Sample Preparation Enhancement:

  • Antigen retrieval optimization (citrate buffer pH 6.0 versus EDTA buffer pH 9.0)

  • Detergent selection affects membrane protein accessibility (Triton X-100 versus NP-40)

  • Protein enrichment through immunoprecipitation before detection

• Reduction of Non-Specific Background:

  • Pre-adsorption of antibodies against tissue/cell lysates lacking PRDM10

  • Optimization of blocking solutions (BSA concentration, addition of normal serum)

  • Including casein or fish gelatin as alternative blocking agents

• Technical Parameter Optimization:

  • Temperature effects (4°C incubation improves specificity but reduces binding kinetics)

  • Extended incubation times at lower antibody concentrations improve signal-to-noise ratio

  • Specialized detection plates/membranes with higher protein binding capacity

• Data Acquisition Optimization:

  • Extended exposure times with low-noise detection systems

  • Digital signal averaging across multiple acquisitions

  • Deconvolution algorithms for improved signal resolution

These approaches collectively enhance detection sensitivity while maintaining specificity, enabling reliable detection of even low-abundance PRDM10 protein in complex biological samples.

How do PRDM10 antibodies contribute to understanding embryonic development pathways?

PRDM10 antibodies have been instrumental in elucidating critical developmental pathways:

• Developmental Expression Profiling:

  • Immunohistochemistry with PRDM10 antibodies enables spatiotemporal mapping of expression

  • PRDM10 expression patterns correlate with critical developmental transitions

  • Comparative analysis across species reveals evolutionary conservation of function

• Mechanistic Pathway Analysis:

  • ChIP-seq using PRDM10 antibodies has identified direct target genes including Eif3b, a key translation initiation factor

  • This established PRDM10's role in maintaining global translation essential for mESC survival

  • Functional validation of these targets reveals developmental checkpoint mechanisms

• Protein-Protein Interaction Networks:

  • Immunoprecipitation with PRDM10 antibodies followed by mass spectrometry identifies interacting partners

  • These interactions reveal integration with broader developmental regulatory networks

  • Temporal dynamics of interactions correlate with developmental stage transitions

• Conditional Knockout Validation:

  • PRDM10 antibodies confirm protein depletion in conditional knockout models

  • This enables precise correlation between protein loss and developmental phenotypes

  • Rescue experiments with mutant PRDM10 variants help map structure-function relationships

Through these applications, PRDM10 antibodies have helped establish that PRDM10 acts as a transcription factor binding to promoters of target genes, regulating their expression, and maintaining translational processes essential for embryonic development .

What insights have PRDM10 antibodies provided about its role in transcriptional regulation?

PRDM10 antibodies have enabled several key discoveries regarding its transcriptional regulatory functions:

• Genomic Binding Profile Characterization:

  • ChIP-seq with validated PRDM10 antibodies identified 528 reproducible binding peaks in mouse embryonic stem cells

  • Analysis revealed that 70.8% of PRDM10 binding sites reside within 1 kb upstream or overlapping with gene transcriptional start sites (TSSs)

  • Only 9.8% of binding sites map to intergenic regions, confirming predominant promoter-proximal activity

• Chromatin Association Patterns:

  • PRDM10 binding strongly associates with regions of transcriptionally active chromatin

  • These regions are characterized by specific histone modifications: H3K4me3, H3K4me1, and H3K36me3

  • This pattern distinguishes PRDM10 from repressive transcription factors

• DNA Motif Recognition:

  • De novo motif discovery from ChIP-seq data identified a consensus sequence enriched within PRDM10 binding sites

  • This motif showed central enrichment in PRDM10 peaks and strong sequence conservation

  • Mutational analysis confirmed the functional relevance of this motif for PRDM10-mediated transcriptional activation

• Domain-Function Relationships:

  • Analysis of PRDM10 deletion mutants demonstrated that the zinc finger DNA-binding domain (ZF-DBD) is essential but insufficient for transcriptional activation

  • This indicates a requirement for additional effector domains in the complete transcriptional regulatory function

  • The PR domain's specific biochemical function in PRDM10 remains under investigation

These findings collectively establish PRDM10 as a sequence-specific transcription factor with direct DNA binding capabilities and activator functions at proximal promoter regions.

How can researchers integrate PRDM10 antibody data with other -omics approaches for comprehensive pathway analysis?

Integrating PRDM10 antibody-generated data with other -omics approaches enables systems-level understanding of its biological functions:

• Multi-omics Data Integration Framework:

  • ChIP-seq data from PRDM10 antibodies identifies direct DNA binding sites

  • RNA-seq following PRDM10 manipulation reveals transcriptional consequences

  • Proteomics approaches identify protein interaction networks

  • Integration through computational platforms (Cytoscape, STRING) reveals emergent properties

• Network Analysis Approaches:

  • PRDM10 binding data can be incorporated into gene regulatory networks

  • Motif co-occurrence analysis identifies cooperative transcription factor complexes

  • Network perturbation analysis predicts critical nodes and potential intervention points

  • Temporal network dynamics reveal developmental stage-specific functions

• Cross-platform Validation Strategies:

  • PRDM10 ChIP-seq peaks can be validated with CUT&RUN or CUT&Tag approaches

  • PRDM10-regulated genes identified by RNA-seq can be confirmed with RT-qPCR

  • Protein interactions detected by co-IP can be validated with proximity labeling approaches

  • Functional significance can be confirmed with targeted genetic manipulations

• Integrated Data Visualization and Analysis:

  • Genome browsers with integrated tracks for binding, expression, and chromatin state

  • Heatmap clustering of multi-dimensional data reveals co-regulated gene modules

  • Principal component analysis identifies major sources of variation across datasets

  • Pathway enrichment tools connect observed changes to biological processes

This integrative approach has revealed PRDM10's critical role in regulating translation initiation factors, particularly Eif3b, which maintains global translation essential for mouse embryonic stem cell survival and early embryonic development .

What emerging applications of PRDM10 antibodies are advancing developmental biology research?

PRDM10 antibodies are enabling several exciting new research directions in developmental biology:

• Single-cell Protein Profiling:

  • Adapting PRDM10 antibodies for CyTOF and CITE-seq applications

  • Correlating PRDM10 levels with developmental trajectories at single-cell resolution

  • Mapping cell-specific PRDM10 functions within heterogeneous developing tissues

• Spatial Transcriptomics Integration:

  • Combining immunohistochemistry with PRDM10 antibodies with spatial transcriptomics

  • Creating spatially-resolved maps of PRDM10 activity and target gene expression

  • Understanding regional specialization of PRDM10 function during development

• Developmental Chromatin Architecture:

  • Using PRDM10 antibodies in ChIA-PET or HiChIP applications

  • Mapping long-range chromatin interactions mediated by PRDM10

  • Understanding 3D genome reorganization during developmental transitions

• Live-cell Dynamics:

  • Developing antibody-based biosensors for real-time PRDM10 activity monitoring

  • Tracking PRDM10 localization during differentiation and cell cycle progression

  • Correlating dynamic PRDM10 behavior with developmental decision points

These emerging approaches promise to reveal new dimensions of PRDM10 biology and its role in coordinating developmental processes through transcriptional and translational regulation mechanisms.

What methodological advances are needed to address current limitations in PRDM10 antibody applications?

Despite significant progress, several methodological challenges remain in PRDM10 antibody applications:

• Isoform-Specific Detection Improvements:

  • Development of antibodies that specifically recognize different PRDM10 isoforms

  • Enhanced epitope mapping to distinguish closely related sequences

  • Validation strategies that definitively confirm isoform specificity

• Post-Translational Modification Monitoring:

  • Generation of antibodies specific for phosphorylated, SUMOylated, or otherwise modified PRDM10

  • Development of protocols that preserve labile modifications during sample preparation

  • Quantitative approaches to determine modification stoichiometry

• Improved Chromatin Immunoprecipitation Efficiency:

  • Development of recombinant antibody fragments with enhanced chromatin accessibility

  • Optimization of crosslinking conditions specific for zinc finger proteins

  • Reduction of background binding through engineered antibody modifications

• Enhanced Quantification Methods:

  • Development of absolute quantification standards for PRDM10 protein levels

  • Improved normalization approaches for cross-sample comparisons

  • Higher-sensitivity detection methods for tissues with low PRDM10 expression

Addressing these methodological challenges will further enhance our understanding of PRDM10's diverse functions in development and disease contexts.

How might PRDM10 antibody research contribute to understanding developmental disorders?

PRDM10 antibody research has significant potential to illuminate mechanisms underlying developmental disorders:

• Developmental Disorder Biomarker Identification:

  • Profiling PRDM10 levels and localization in patient-derived samples

  • Correlating PRDM10 dysfunction with specific developmental phenotypes

  • Identifying downstream molecular signatures of PRDM10 dysregulation

• Patient-Specific iPSC Models:

  • Using PRDM10 antibodies to validate patient-derived induced pluripotent stem cell models

  • Monitoring PRDM10 during differentiation of patient cells versus controls

  • Identifying cell type-specific vulnerabilities to PRDM10 dysfunction

• Therapeutic Target Validation:

  • Employing PRDM10 antibodies to monitor treatment responses in preclinical models

  • Validating small molecule or genetic interventions targeting PRDM10 pathways

  • Developing companion diagnostics for stratification of developmental disorder subtypes

• Mechanistic Disease Classification:

  • Using PRDM10 pathway signatures to develop molecular classifications of developmental disorders

  • Distinguishing primary from secondary effects in complex developmental syndromes

  • Identifying convergent pathways across seemingly disparate developmental disorders