INTS10 Antibody

What is INTS10 and why is it significant for transcription research?

INTS10 functions as a subunit of the Integrator complex, which associates with the C-terminal domain of RNA polymerase II large subunit (POLR2A) and mediates 3′-end processing of small nuclear RNAs such as U1 . The Integrator complex is critical for regulating transcription, particularly in establishing and modulating paused RNA polymerase II. INTS10 specifically interacts with other Integrator subunits including INTS13, INTS14, and the recently characterized INTS15 to form the functionally important "Arm module" of the complex . Studying INTS10 provides insight into fundamental mechanisms of transcriptional regulation and RNA processing in eukaryotic cells.

What species reactivity should I consider when selecting an INTS10 antibody?

Commercial INTS10 antibodies demonstrate varying degrees of cross-reactivity across species. Based on sequence homology analysis, antibodies raised against human INTS10 often show high cross-reactivity with mouse (93-96% sequence identity) and rat (97-100% sequence identity) orthologs . Many commercial antibodies are also predicted to recognize INTS10 in other mammals including cow (100%), dog (86%), guinea pig (100%), rabbit (93%), and horse (93%) . When selecting an antibody for your experimental system, verify the vendor's validation data in your species of interest, as sequence conservation does not always translate to equivalent antibody performance across species.

What epitope regions are most commonly targeted in commercial INTS10 antibodies?

Commercial INTS10 antibodies typically target either the C-terminal region or internal domains of the protein. C-terminal directed antibodies often recognize epitopes within amino acids 516-553 , while others target larger internal segments (e.g., AA 451-710) . The choice of epitope region can significantly impact experimental outcomes, particularly when studying protein interactions. For instance, antibodies targeting the C-terminal helical repeats might interfere with the INTS10-INTS14 interaction, as this interface involves residues in this region (e.g., E633, E634) . Conversely, N-terminal directed antibodies could potentially disrupt INTS10-INTS15 binding, which depends on the first N-terminal helical repeat (residues 1-37) of INTS10 .

How should I validate the specificity of an INTS10 antibody for my research?

A comprehensive validation approach should include multiple techniques:

• Western blot analysis: Verify single band detection at the expected molecular weight (~70 kDa for human INTS10). Include positive and negative control lysates.

• Knockdown/knockout controls: Compare antibody signal between wild-type cells and those with INTS10 depleted via RNAi or CRISPR-Cas9. A specific antibody will show substantially reduced signal in knockdown/knockout samples.

• Immunoprecipitation followed by mass spectrometry: Perform IP-MS as described in Offley et al., using ~1.5 mg nuclear extract per IP with 4 μg antibody and appropriate magnetic beads . Specific antibodies should enrich INTS10 and known interacting partners like INTS13, INTS14, and INTS15.

• Recombinant protein detection: Test the antibody against purified recombinant INTS10 to confirm direct recognition of the target protein.

• Structural-functional validation: If studying INTS10 interactions, validate antibody compatibility with your experimental design by ensuring it doesn't target interaction interfaces. For example, antibodies targeting residues 1-37 might disrupt INTS10-INTS15 interactions, while those recognizing the C-terminal region could affect INTS10-INTS14 binding .

What is the optimal protocol for immunoprecipitation experiments with INTS10 antibodies?

Based on the protocols described in the literature, the following methodology is recommended for INTS10 immunoprecipitation:

• Sample preparation: Prepare nuclear extracts from your cells of interest. Use approximately 1.5 mg of nuclear extract per IP.

• Buffer composition: Dilute samples in Co-IP buffer containing 20 mM Tris pH 8.0, 100 mM NaCl, 0.1% NP-40, and protease inhibitors .

• Antibody binding: For each IP, use 4 μg of INTS10 antibody (or 10 μL of serum) coupled to 30 μL of Dynabeads Protein A or Protein G .

• Incubation conditions: Incubate the IP reactions at 4°C for 2 hours with rotation.

• Washing procedure: Wash beads three times with Co-IP buffer, followed by an additional wash with 0.05% NP-40 in 1X PBS .

• Elution method: Elute bound proteins with 0.1 M glycine pH 3.0, then neutralize the eluate pH with 1 M Tris base pH 11.0 .

• Analysis: Analyze the eluates by SDS-PAGE/Western blot or prepare for LC-MS/MS to identify co-immunoprecipitated proteins.

Include appropriate controls such as IgG control antibodies and input samples to assess non-specific binding and IP efficiency.

What applications are most suitable for INTS10 antibody research?

Commercial INTS10 antibodies have been validated for multiple applications with varying degrees of success:

When selecting an antibody, prioritize those validated for your specific application and verify the validation data provided by the manufacturer.

How can I use INTS10 antibodies to investigate Integrator complex assembly and dynamics?

INTS10 antibodies can be powerful tools for studying Integrator complex organization through several sophisticated approaches:

• Sequential immunoprecipitation: Perform tandem IP experiments (first with INTS10 antibodies, then with antibodies against other Integrator subunits) to isolate specific subcomplexes and determine the hierarchical assembly of the complex.

• Proximity-dependent labeling: Combine INTS10 antibodies with techniques like BioID or APEX to map the spatial organization of INTS10 relative to other Integrator components in living cells.

• Crosslinking followed by IP-MS: As demonstrated by Offley et al., crosslinking with BS3 followed by IP-MS can identify direct protein-protein interactions involving INTS10 . This approach confirmed the predicted INTS10-INTS15 interface.

• Structure-guided mutagenesis with antibody detection: Create point mutations at predicted interaction interfaces (e.g., W28P/L29P or E633A/E634A in INTS10) and use antibodies to track how these mutations affect complex assembly . For example, INTS10 W28P/L29P mutations disrupt INTS15 binding while maintaining INTS13/14 interactions.

• ChIP-seq with INTS10 antibodies: Map INTS10 genomic binding sites and compare with the localization patterns of other Integrator subunits to identify both common and subunit-specific recruitment sites.

How can I study the specific interaction between INTS10 and INTS15 using antibodies?

The INTS10-INTS15 interaction represents a critical structural feature of the Integrator complex Arm module. To study this interaction:

• Co-immunoprecipitation with structural insights: Use INTS10 antibodies that don't target the N-terminal helical repeat (residues 1-37), as this region forms the interface with INTS15 . Perform reciprocal IPs with INTS15 antibodies to confirm interaction.

• Interface mutagenesis validation: Generate INTS10 mutants (W28P/L29P or deletion of residues 1-37) or INTS15 mutants (L384A/L387A) that disrupt their interaction . Use antibodies against unmodified regions to detect these proteins in IP experiments.

• Quantitative proteomics approach: Perform differential quantitative proteomics of affinity-purified wild-type versus mutant INTS10 as described by Offley et al. . This can reveal how disrupting specific interfaces affects recruitment of other complex components.

• Chromatin recruitment analysis: Use ChIP with INTS10 and INTS15 antibodies to determine if these proteins are co-recruited to the same genomic loci, particularly at active genes or enhancers where INTS15 has been shown to localize .

• Structural validation: Combine antibody-based approaches with other structural techniques like negative-stain EM to validate the predicted L-shaped arrangement of the INTS10-INTS15 dimer .

What approaches can identify novel INTS10 interaction partners beyond known Integrator subunits?

To discover new INTS10 interacting proteins:

• Unbiased IP-MS screening: Perform large-scale immunoprecipitation with INTS10 antibodies followed by mass spectrometry analysis. Compare against IgG controls to identify specific interactions.

• Differential interactome analysis: Compare the interactome of wild-type INTS10 with mutants that disrupt known interactions. For example, the E633A/E634A INTS10 mutant showed altered interaction with ZNF655, suggesting additional DNA-binding proteins may interact with the Arm module .

• Cell-state dependent interactome: Analyze INTS10 interactions under different cellular conditions (e.g., before and after EGF treatment) to identify dynamic or context-specific interactions .

• Chromatin-focused analysis: Perform ChIP-MS to identify proteins that co-occupy genomic loci with INTS10, potentially identifying transcription factors that recruit INTS10 to specific sites.

• Proximity labeling approaches: Use BioID or APEX2 fused to INTS10 coupled with antibody validation to identify proteins in close proximity to INTS10 in living cells.

Why might I observe multiple bands when using INTS10 antibodies in Western blot?

Multiple bands in Western blots using INTS10 antibodies could result from several factors:

• Post-translational modifications: INTS10 may undergo phosphorylation or other modifications that alter its migration pattern.

• Alternative splicing: Check databases for reported splice variants of INTS10 that might explain additional bands.

• Proteolytic processing: INTS10 might undergo partial degradation during sample preparation. Include protease inhibitors and minimize sample processing time.

• Cross-reactivity: The antibody may recognize proteins with similar epitopes. Validate with negative controls (knockdown/knockout samples) or competition experiments with the immunizing peptide.

• Sample preparation issues: Optimize cell lysis conditions and protein denaturation to ensure complete extraction and denaturation of INTS10.

To address this issue, compare results with different antibodies recognizing distinct INTS10 epitopes, and validate any unexpected bands through targeted approaches like immunoprecipitation followed by mass spectrometry.

How should I interpret contradictory results between different commercial INTS10 antibodies?

Discrepancies between different INTS10 antibodies could arise from:

• Epitope availability: Different antibodies recognize distinct epitopes that may be differentially accessible in various experimental contexts. Map the epitopes of each antibody relative to known functional domains and interaction interfaces of INTS10.

• Post-translational modifications: Some antibodies may be sensitive to modifications near their epitopes. For example, an antibody targeting the C-terminal region might be affected by phosphorylation events that regulate INTS10-INTS14 interaction .

• Conformational differences: INTS10 may adopt different conformations depending on its interaction partners. The availability of epitopes could change when INTS10 interacts with INTS15 versus INTS14 .

• Antibody quality variation: Different production lots can exhibit variable quality. Always include positive controls validated with each antibody.

• Context-dependent interactions: INTS10 forms different subcomplexes in different cellular contexts. For instance, INTS15 has been identified at genomic loci where other Integrator subunits are absent .

To resolve contradictions, perform validation experiments using multiple antibodies in parallel, and incorporate orthogonal approaches like mass spectrometry to confirm protein identity and interactions.

What controls should I include when using INTS10 antibodies for chromatin immunoprecipitation (ChIP) experiments?

For reliable INTS10 ChIP experiments, include these essential controls:

• Input DNA: Always process an aliquot of starting chromatin material before immunoprecipitation.

• IgG control: Use matched isotype control antibodies to assess non-specific binding.

• Positive genomic loci: Include primers for regions where INTS10 is expected to bind, such as U snRNA genes or EGF-responsive genes where Integrator recruitment has been demonstrated .

• Negative genomic loci: Include primers for regions not expected to recruit INTS10, such as inactive genes or intergenic regions.

• Knockdown/knockout validation: Perform ChIP in cells with reduced INTS10 expression to confirm signal specificity.

• Cooperative binding controls: Include ChIP for other Integrator subunits (e.g., INTS11, INTS15) to confirm co-occupancy. Research shows INTS10 and INTS15 are recruited alongside RNA polymerase II at EGF-responsive genes .

• Context-specific controls: If studying specific cellular conditions like EGF treatment, include appropriate time-course samples, as INTS10 recruitment can be dynamically regulated .

Proper normalization and quantification are essential for interpreting ChIP data accurately. Consider performing ChIP-seq rather than ChIP-qPCR to obtain genome-wide binding profiles when possible.

How might emerging technologies enhance INTS10 antibody applications in research?

Several cutting-edge approaches could advance INTS10 research:

• Integrative structural biology: Combining antibody-based techniques with AlphaFold2 predictions and cryo-EM has already yielded insights into INTS10-INTS15 interactions . This integrative approach can be expanded to study the entire Integrator complex architecture.

• Single-molecule imaging: Using fluorescently labeled INTS10 antibodies for super-resolution microscopy could reveal the spatial organization and dynamics of Integrator complexes in single cells.

• CUT&Tag and CUT&RUN: These techniques offer advantages over traditional ChIP for mapping genomic binding sites of INTS10 with higher sensitivity and reduced background.

• Proximity proteomics in specific nuclear compartments: Combining antibodies with proximity labeling in specific nuclear domains could reveal context-specific INTS10 interactions.

• Single-cell approaches: Adapting INTS10 antibodies for single-cell proteomics or CyTOF could reveal cell-to-cell variability in Integrator complex composition and abundance.

These emerging technologies, when combined with well-validated INTS10 antibodies, promise to deepen our understanding of how the Integrator complex contributes to transcriptional regulation across different cellular contexts.