PRDM10 Antibody

What is PRDM10 and what cellular functions does it serve?

PRDM10 is a member of the PRDM (PR/SET domain-containing) family of transcriptional regulators characterized by an N-terminal PR domain (similar to the SET domain found in histone methyltransferases) and multiple C2H2-type zinc finger motifs. Functionally, PRDM10 acts as a sequence-specific transcription factor involved in:

• Regulation of gene expression and chromatin remodeling

• Maintenance of embryonic stem cell homeostasis

• Essential processes during pre-implantation embryonic development

• Regulation of global translation through its downstream target Eif3b

• Possible role as a gene repressor involved in tissue differentiation

Research has demonstrated that PRDM10 knockout in mouse embryonic stem cells (mESCs) results in severe growth inhibition, highlighting its essential nature in early development .

What are the key technical specifications of commercial PRDM10 antibodies?

When selecting a PRDM10 antibody for research applications, consider these key specifications based on commercial offerings:

Most commercially available antibodies require optimization for specific experimental systems to obtain optimal results .

How can I validate the specificity of a PRDM10 antibody for my research?

Proper validation of PRDM10 antibody specificity should include multiple complementary approaches:

• Positive and negative control samples: Test the antibody on cells or tissues known to express (e.g., HEK-293 cells, mouse testis tissue) or not express PRDM10.

• Knockdown/knockout validation: Compare antibody reactivity between wild-type samples and those with PRDM10 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9). The signal should be significantly reduced or absent in knockdown/knockout samples.

• Blocking peptide competition: Pre-incubate the antibody with its immunizing peptide before application to the sample. A specific antibody will show diminished or absent signal.

• Multiple antibody verification: Use more than one antibody targeting different epitopes of PRDM10 to confirm consistent detection patterns.

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight (120-150 kDa for PRDM10) .

What are the optimal conditions for Western blot detection of PRDM10?

For successful Western blot detection of PRDM10 (observed MW: 120-150 kDa), implement the following optimized protocol:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

  • Heat samples at 95°C for 5 minutes in reducing Laemmli buffer

• Gel electrophoresis:

  • Use 6-8% SDS-PAGE gels due to PRDM10's high molecular weight

  • Run at lower voltage (80-100V) to improve resolution of high MW proteins

• Transfer conditions:

  • Wet transfer is recommended for large proteins like PRDM10

  • Transfer at 30V overnight at 4°C or 100V for 2 hours with cooling

  • Use 0.45 μm PVDF membrane (not nitrocellulose) for better retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Incubate with primary antibody at 1:500-1:2000 dilution overnight at 4°C

  • Wash extensively (4-5 times, 5 minutes each) with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour

• Detection:

  • Use enhanced chemiluminescence with longer exposure times (1-5 minutes)

  • Confirm specificity using positive control samples (HEK-293 cells)

How can I optimize immunoprecipitation protocols for PRDM10?

For successful immunoprecipitation of PRDM10 from complex samples:

• Lysis optimization:

  • Use gentle lysis buffers (e.g., 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA) with protease inhibitors

  • Include 10-20 mM N-ethylmaleimide to preserve potential SUMOylation

  • Lyse cells on ice for 30 minutes with gentle agitation

• Antibody amount:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Pre-clear lysate with Protein A/G beads for 1 hour before adding antibody

• Immunoprecipitation conditions:

  • Incubate lysate with antibody overnight at 4°C under gentle rotation

  • Add pre-washed Protein A/G beads for 2-4 hours

  • Perform at least 4 washes with lysis buffer

• Elution strategies:

  • For co-IP analysis: use gentle elution with antibody-specific peptide

  • For downstream mass spectrometry: elute with 0.1 M glycine (pH 2.5) and immediately neutralize

• Verification:

  • Include IgG control immunoprecipitation

  • Verify results with Western blot using a different PRDM10 antibody than used for IP

  • Mouse testis tissue has been validated for successful PRDM10 IP

What considerations are important for immunohistochemical detection of PRDM10?

For reliable immunohistochemical (IHC) detection of PRDM10 in tissue samples:

• Fixation optimization:

  • Formalin-fixed paraffin-embedded (FFPE) tissues require antigen retrieval

  • Optimal antigen retrieval: citrate buffer (pH 6.0) for 20 minutes at 95°C

  • Consider testing both FFPE and frozen sections for comparison

• Antibody concentration:

  • Initial testing should cover a range of dilutions (1:50 to 1:500)

  • Include positive controls (tissues known to express PRDM10)

  • Include negative controls (primary antibody omission and non-expressing tissues)

• Signal enhancement and background reduction:

  • Use biotin-free detection systems to reduce background

  • Employ signal amplification for low-abundance detection

  • Block endogenous peroxidase (3% H₂O₂) and biotin if relevant

• Interpretation guidance:

  • PRDM10 expression is primarily nuclear with possible nucleolar enrichment

  • In soft tissue sarcomas, positive staining is observed in approximately 19% of cases

  • PRDM10-positive tumors often display myxoid changes and multinucleated giant cells

• IHC scoring method:

  • Score staining intensity (0-3) and percentage of positive cells

  • Calculate H-score (intensity × percentage) for semi-quantitative analysis

  • Compare with clinicopathological features as PRDM10 expression correlates with specific tumor characteristics

How can PRDM10 antibodies be used to investigate protein-protein interactions?

For investigating PRDM10 protein-protein interactions, implement these advanced approaches:

• Co-immunoprecipitation (Co-IP):

  • Use 3-5 μg of PRDM10 antibody per 1 mg protein lysate

  • Perform stringency optimization with different salt concentrations (150-500 mM NaCl)

  • Consider crosslinking with DSP or formaldehyde to capture transient interactions

  • Analyze by Western blot or mass spectrometry for interacting partners

• Proximity ligation assay (PLA):

  • Combine PRDM10 antibody with antibodies against suspected interaction partners

  • Use species-specific secondary antibodies with oligonucleotide probes

  • Signal amplification enables visualization of protein interactions in situ

  • Quantify interaction events per cell to measure interaction strength

• ChIP-seq combined with protein complex analysis:

  • Perform chromatin immunoprecipitation using PRDM10 antibody

  • Identify DNA binding sites and associated protein complexes

  • Re-ChIP (sequential ChIP) to identify co-occupancy with other factors

  • Investigate PRDM10's reported role as a sequence-specific transcription factor

• BioID or APEX proximity labeling:

  • Generate PRDM10-BioID/APEX fusion constructs

  • Use antibodies to validate correct fusion protein expression

  • Compare biotinylated proteins with PRDM10 antibody immunoprecipitation results

  • Focus on interaction partners in translation regulation pathways, particularly related to EIF3B

What approaches can resolve conflicting PRDM10 antibody results in different experimental systems?

When facing inconsistent PRDM10 antibody results across different experimental systems, implement this systematic troubleshooting strategy:

• Epitope mapping and antibody selection:

  • Determine the exact epitope recognized by each antibody

  • Select antibodies targeting different epitopes of PRDM10

  • Consider potential isoform differences that may affect epitope accessibility

  • Verify if post-translational modifications could mask epitopes

• Cell/tissue-specific expression analysis:

  • Compare PRDM10 mRNA levels (qRT-PCR/RNA-seq) with protein detection

  • Analyze alternative splicing patterns in different tissues

  • Consider cell-type specific regulation of PRDM10 expression

  • Use fluorescent-tagged PRDM10 expression constructs as validation tools

• Sample preparation optimization:

  • Test multiple lysis/extraction protocols to ensure complete protein solubilization

  • Optimize detergent types and concentrations for membrane-associated fractions

  • Test denaturation conditions (temperature, time, reducing agents)

  • Consider native vs. denaturing conditions for epitope preservation

• Cross-validation strategies:

  • Combine multiple detection methods (Western blot, ICC/IF, IP-MS)

  • Perform siRNA/CRISPR knockdown controls in each experimental system

  • Use recombinant PRDM10 fragments as positive controls

  • Integrate computational predictions with experimental observations

How can I investigate PRDM10's role in transcriptional regulation using antibody-based techniques?

To explore PRDM10's function as a transcription factor, employ these antibody-dependent methodologies:

• Chromatin Immunoprecipitation (ChIP) and ChIP-seq:

  • Optimize crosslinking conditions (1% formaldehyde for 10-15 minutes)

  • Sonicate chromatin to 200-500 bp fragments

  • Use 3-5 μg PRDM10 antibody per ChIP reaction

  • Include IgG control and positive control for a known PRDM10 target (Eif3b)

  • Analyze enriched regions for common DNA binding motifs

  • Focus analysis on genes involved in translation regulation

• CUT&RUN or CUT&Tag alternatives:

  • For limited samples, use these more sensitive techniques

  • Requires optimization of antibody concentration (typically 0.5-1 μg)

  • Provides higher signal-to-noise ratio than traditional ChIP

  • Compare binding profiles with published PRDM10 binding sites

• Sequential ChIP (Re-ChIP):

  • Investigate co-occupancy with other transcription factors

  • First ChIP with PRDM10 antibody, then use antibodies against suspected partners

  • Focus on components of the translation initiation complex

  • Requires careful optimization of elution conditions between ChIPs

• Transcription factor activity assays:

  • Use reporter constructs containing PRDM10 binding sites

  • Perform antibody-mediated inhibition in nuclear extracts

  • Combine with PRDM10 overexpression/knockdown studies

  • Evaluate direct effects on Eif3b expression and global translation

• Genome-wide approaches:

  • Integrate ChIP-seq with RNA-seq following PRDM10 manipulation

  • Use antibody-based RNA immunoprecipitation (RIP) to identify PRDM10-associated transcripts

  • Perform PRDM10 ChIP-mass spectrometry to identify co-factors

  • Focus on early embryonic development contexts where PRDM10 is essential

How can PRDM10 antibodies be utilized in sarcoma classification research?

For investigating PRDM10's potential as a diagnostic marker in soft tissue sarcomas:

• IHC optimization for diagnostic applications:

  • Use standardized protocols on tissue microarrays

  • Establish scoring criteria: intensity (0-3) and percentage of positive cells

  • Compare with standard sarcoma markers

  • Focus on undifferentiated pleomorphic sarcomas (UPS)

• Clinicopathological correlation analysis:

  • PRDM10 expression shows significant correlation with:

    • Myxoid changes (p = 0.017)

    • Multinucleated giant cells (p = 0.034)

    • Surgical margin status (p = 0.032)

  • PRDM10-positive tumors are more likely to have well-circumscribed margins

  • Approximately 19% of soft tissue sarcomas show PRDM10 positivity by IHC

• Detection of PRDM10 fusion transcripts:

  • Use antibodies to screen for potential fusion proteins

  • Combine with FISH or RT-PCR for fusion transcript detection

  • Focus on low-grade undifferentiated pleomorphic sarcomas

  • Correlate antibody staining patterns with fusion status

• Prognostic significance assessment:

  • Perform survival analysis stratified by PRDM10 expression

  • Compare with standard prognostic parameters

  • Evaluate potential for therapeutic targeting

  • Establish tissue collection and staining SOPs for clinical research

What methodological approaches can detect PRDM10 in liquid biopsies for cancer research?

For developing PRDM10-based liquid biopsy approaches:

• Extracellular vesicle (EV) isolation and analysis:

  • Isolate EVs from patient serum/plasma using ultracentrifugation or size-exclusion chromatography

  • Validate EV isolation with EV markers (CD63, CD9)

  • Use PRDM10 antibodies for Western blot analysis of EV content

  • Estimate sensitivity limits through dilution experiments with recombinant standards

• Circulating tumor cell (CTC) detection:

  • Isolate CTCs using immunomagnetic separation

  • Perform immunocytochemistry with PRDM10 antibodies

  • Establish multi-color panels including PRDM10 and other sarcoma markers

  • Compare PRDM10 expression in CTCs vs. primary tumors

• Circulating tumor DNA (ctDNA) correlation:

  • Correlate PRDM10 protein expression in tumors with ctDNA markers

  • Establish protocols for combined protein and nucleic acid analysis

  • Develop integrated liquid biopsy approaches

  • Study relationship between PRDM10 mutations/fusions and protein expression

• Multiplex assay development:

  • Design antibody panels including PRDM10 for multiplex analysis

  • Optimize simultaneous detection of multiple markers

  • Validate with digital pathology quantification methods

  • Establish quality control metrics for clinical research applications

How can PRDM10 antibodies be utilized to study its role in embryonic development?

For investigating PRDM10's essential function in early embryonic development:

• Temporal-spatial expression analysis:

  • Perform immunohistochemistry on staged embryonic tissues

  • Use confocal microscopy to determine subcellular localization

  • Combine with lineage markers to identify cell-type specific expression

  • Track PRDM10 expression during pre-implantation development

• Conditional knockout model analysis:

  • Use PRDM10 antibodies to validate knockout efficiency

  • Perform IHC and Western blot to confirm protein depletion

  • Study phenotypic consequences in embryonic stem cells

  • Investigate defects in global translation processes

• Protein-protein interaction networks during development:

  • Perform stage-specific co-immunoprecipitation experiments

  • Identify developmental stage-specific interaction partners

  • Focus on interactions with translation initiation factors

  • Study the relationship between PRDM10 and EIF3B in developmental contexts

• Genome editing validation:

  • Use antibodies to validate CRISPR/Cas9 knockout efficiency

  • Perform rescue experiments with mutant PRDM10 constructs

  • Study domain-specific functions through deletion constructs

  • Investigate the severe growth inhibition phenotype observed in PRDM10-deficient mESCs

What methodological considerations are important when studying PRDM10's role in translation regulation?

To investigate PRDM10's function in regulating global translation through EIF3B:

• Combined ChIP and translation assays:

  • Use PRDM10 antibodies for ChIP at the Eif3b promoter

  • Quantify EIF3B protein levels by Western blot

  • Correlate PRDM10 binding with Eif3b expression levels

  • Perform polysome profiling to assess translation efficiency

• Metabolic labeling optimization:

  • Use puromycin incorporation (SUnSET method) to measure translation rates

  • Combine with PRDM10 antibody staining to correlate expression with translation activity

  • Perform time-course experiments following PRDM10 manipulation

  • Compare results in wild-type and PRDM10-depleted cells

• Ribosome profiling integration:

  • Correlate PRDM10 ChIP-seq data with ribosome profiling results

  • Analyze translation efficiency of specific mRNA subsets

  • Focus on genes involved in embryonic development

  • Study the global translation defects in PRDM10-deficient cells

• Proximity-based approaches:

  • Use PRDM10 antibodies for proximity ligation assays with translation factors

  • Investigate physical interactions with EIF3B and other translation initiation factors

  • Perform microscopy-based co-localization studies

  • Assess PRDM10 association with translating ribosomes

How can single-cell techniques be combined with PRDM10 antibodies for developmental studies?

For applying cutting-edge single-cell approaches to PRDM10 research:

• Single-cell Western blot optimization:

  • Adapt PRDM10 antibody protocols for microfluidic single-cell Western blot

  • Optimize lysis conditions and antibody concentrations

  • Validate with recombinant standards and controls

  • Correlate with single-cell RNA-seq for multi-omics integration

• Mass cytometry (CyTOF) panel development:

  • Conjugate PRDM10 antibodies with rare earth metals

  • Design panels including developmental markers

  • Optimize signal-to-noise ratio and staining protocols

  • Apply to early embryo development studies

• Spatial transcriptomics integration:

  • Combine PRDM10 antibody staining with spatial transcriptomics

  • Correlate protein expression with spatially-resolved transcription

  • Focus on pre-implantation embryos and stem cell niches

  • Investigate spatial relationship between PRDM10 and its target genes

• Live-cell imaging approaches:

  • Use fluorescently-labeled antibody fragments for live imaging

  • Validate specificity against PRDM10-fluorescent protein fusions

  • Track PRDM10 dynamics during cell division and differentiation

  • Correlate with translation activity markers in real-time