INTS10 Antibody, HRP conjugated

What is INTS10 and what cellular functions does it participate in?

INTS10 (Integrator complex subunit 10, also known as Int10 or C8orf35) is a protein component of the Integrator complex that plays critical roles in epigenetic regulation and nuclear signaling pathways . INTS10 forms a functional module with INTS13 and INTS14 within the Integrator complex . Recent research has identified that this module also includes INTS15, forming a stable tetrameric arrangement now referred to as the "Arm module" . The Integrator complex is involved in multiple nuclear processes, including transcriptional regulation and RNA processing. INTS10 specifically appears to localize at active enhancers and certain cis-regulatory genomic elements, suggesting its importance in transcriptional control mechanisms .

What are the key specifications of commercially available INTS10 antibodies with HRP conjugation?

The HRP-conjugated INTS10 antibody is a polyclonal antibody raised in rabbits against recombinant human Integrator complex subunit 10 protein (specifically amino acids 451-710) . The antibody has the following specifications:

How should the INTS10 antibody be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, INTS10 antibody (HRP conjugated) should be stored at -20°C or -80°C immediately upon receipt . It is crucial to avoid repeated freeze-thaw cycles as this can lead to epitope degradation and loss of binding efficiency. When working with the antibody:

• Aliquot the stock solution into smaller working volumes upon first thawing to minimize future freeze-thaw events

• When thawing, allow the antibody to warm gradually to room temperature

• Mix gently by inversion rather than vortexing to prevent protein denaturation

• Return unused portions to -20°C or -80°C promptly after use

• Note that the antibody is preserved in 50% glycerol with 0.03% Proclin 300, which helps maintain stability during freezing and thawing events

How does INTS10 interact with other components of the Integrator complex, and how can these interactions be studied experimentally?

INTS10 forms distinct molecular interactions within the Integrator complex, primarily through its association with INTS14 and INTS15. The interaction between INTS10 and other subunits can be studied through several experimental approaches:

• Co-immunoprecipitation (Co-IP): INTS10 directly interacts with INTS14, specifically through the VWA domain of INTS14 . This interaction has been experimentally validated where deletion of the INTS14 VWA domain (residues 1-210) completely abolished INTS10 binding while retaining interaction with INTS13 .

• Structural mapping of interaction interfaces: The interaction between INTS10 and INTS15 occurs through a head-to-tail arrangement where the N-terminal helical repeat of INTS10 (residues 1-37) interacts with C-terminal helices of INTS15 . This has been confirmed through:

  • Negative-stain electron microscopy

  • Cross-linking mass spectrometry (XL-MS)

  • Mutagenesis studies where removal of the first helical repeat of INTS10 or introduction of helix-breaking mutations (W28P/L29P) resulted in loss of INTS15 co-purification

• Recombinant protein expression: The INTS10-INTS15 interaction has been validated by co-expression in insect cells, yielding a stable, monodisperse complex that can be analyzed by size-exclusion chromatography .

What experimental considerations are important when using INTS10 antibody (HRP conjugated) for protein detection in complex biological samples?

When using HRP-conjugated INTS10 antibody for protein detection in complex samples, researchers should consider:

• Sample preparation optimization:

  • Nuclear extraction protocols are critical since INTS10 is primarily located in the nucleus as part of the Integrator complex

  • Use suitable lysis buffers that preserve protein-protein interactions if studying INTS10's association with other Integrator subunits

  • Consider salt concentration effects, as high-salt conditions (up to 750 mM KCl) do not disrupt INTS10's association with INTS13 and other subunits

• Antibody specificity validation:

  • Include positive controls using recombinant INTS10 protein

  • Verify specificity through knockdown/knockout approaches

  • Consider pre-adsorption with immunizing peptide to confirm specific binding

• Signal detection optimization:

  • Adjust substrate incubation time based on the expression level of INTS10 in your experimental system

  • Use appropriate blocking agents to minimize non-specific binding

  • Consider signal amplification methods if detecting low-abundance INTS10 in certain cell types

• Experimental controls:

  • Include isotype control antibodies

  • Use cell lines with confirmed INTS10 expression levels (e.g., HEK293T cells have been used successfully for INTS10 studies)

  • Consider phosphatase inhibitors in lysis buffers if studying potential post-translational modifications

What are the recommended approaches for investigating INTS10's role in transcriptional regulation using the HRP-conjugated antibody?

To investigate INTS10's role in transcriptional regulation, researchers can employ the following methodological approaches:

• Chromatin Immunoprecipitation (ChIP) followed by sequencing:

  • INTS10 has been shown to be recruited alongside RNA Polymerase II at EGF-responsive genes

  • ChIP protocol optimization should include:

    • Appropriate crosslinking conditions (typically 1% formaldehyde for 10 minutes)

    • Sonication parameters optimized for nuclear proteins

    • Elution and reversal of crosslinks followed by DNA purification for sequencing

• Integration with transcriptomic analyses:

  • INTS10 involvement in HCC (Hepatocellular Carcinoma) has been studied using TCGA database for transcriptomic analyses

  • Consider comparing INTS10 binding sites with differential gene expression after INTS10 knockdown/knockout

• Enhancer activity studies:

  • INTS10 has been found to localize at active enhancers but not poised enhancers

  • Consider reporter assays with enhancer elements to quantify the impact of INTS10 modulation

  • Use stimulation protocols (e.g., EGF treatment) that have been shown to affect INTS10 recruitment to regulatory elements

What strategies can resolve non-specific binding when using INTS10 antibody (HRP conjugated) in immunodetection applications?

When encountering non-specific binding issues with HRP-conjugated INTS10 antibody, implement these methodological solutions:

• Blocking optimization:

  • Test different blocking reagents (BSA, non-fat dry milk, commercial blockers)

  • Extend blocking time from standard 1 hour to 2-3 hours at room temperature

  • Consider adding 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) to identify optimal signal-to-noise ratio

  • Consider overnight incubation at 4°C instead of shorter incubations at room temperature

• Wash protocol enhancement:

  • Increase wash buffer stringency (consider 0.1-0.5% SDS addition)

  • Extend wash times and increase the number of wash steps

  • Use PBS-T with increasing Tween-20 concentrations (0.05-0.1%)

• Pre-adsorption technique:

  • Pre-incubate the antibody with the immunizing peptide (if available) to confirm specificity

  • Alternatively, use the antibody on lysates from cells with confirmed INTS10 knockdown

How can researchers effectively validate protein-protein interactions between INTS10 and other Integrator subunits?

To validate interactions between INTS10 and other Integrator subunits, employ these methodological approaches:

• Complementary interaction validation techniques:

  • Co-immunoprecipitation with antibodies against different Integrator subunits

  • Proximity ligation assay (PLA) for in situ detection of INTS10 interactions

  • FRET or BiFC for live-cell visualization of interactions

  • In vitro binding assays using recombinant proteins

• Mutagenesis-based validation:

  • Target specific residues based on structural predictions, as done for:

    • INTS10 W28P/L29P mutations that disrupt INTS15 interaction

    • INTS10 E633A and E633A/E634A mutations that disrupt INTS14 recruitment

  • Compare binding efficiencies of wild-type and mutant proteins quantitatively

• Multi-subunit complex reconstitution:

  • Express and purify recombinant INTS10 along with interacting partners (INTS13, INTS14, INTS15)

  • Analyze complex formation using size-exclusion chromatography

  • Apply glycerol gradient centrifugation to assess complex stability, as demonstrated for the INTS13/14/10/15 module

How do post-translational modifications of INTS10 affect its interactions within the Integrator complex, and how can these be studied?

Post-translational modifications (PTMs) of INTS10 may significantly influence its interactions within the Integrator complex. To investigate this:

• Identification of PTM sites:

  • Perform mass spectrometry analysis of immunoprecipitated INTS10 to identify phosphorylation, ubiquitination, or other modifications

  • Use phosphatase or deubiquitinase treatments prior to immunoprecipitation to assess the impact of PTM removal

• Functional analysis of PTMs:

  • Generate site-specific mutants (phospho-mimetic or phospho-deficient) at identified PTM sites

  • Assess the impact on INTS10's interaction with INTS14 and INTS15 through co-IP experiments

  • Evaluate changes in nuclear localization or chromatin association patterns

• Conditional regulation of PTMs:

  • Investigate changes in INTS10 PTMs under different cellular conditions (e.g., differentiation, stress response)

  • Use kinase inhibitors or activators to modulate PTM status and assess effects on INTS10 function

  • Consider the impact of cell cycle progression on INTS10 modification status

What experimental design considerations are important when investigating INTS10's role at enhancers versus promoters?

When studying INTS10's differential roles at enhancers versus promoters, consider these methodological approaches:

• Genomic location analysis:

  • Perform ChIP-seq for INTS10 and compare binding patterns with established enhancer marks (H3K4me1, H3K27ac) versus promoter marks (H3K4me3)

  • Integrate with ATAC-seq to correlate INTS10 binding with chromatin accessibility

  • Consider co-occupancy analysis with other Integrator subunits, particularly INTS13, which has been shown to bind poised enhancers

• Functional distinction experiments:

  • Design reporter assays with enhancer versus promoter elements to assess INTS10's functional impact

  • Use CRISPR-mediated recruitment of INTS10 to specific genomic loci to evaluate context-dependent effects

  • Consider tethering experiments to artificially recruit INTS10 to promoters versus enhancers

• Cell-type specific considerations:

  • INTS10 has been found at active enhancers but not poised enhancers in HL-60 cells differentiating into monocytes/macrophages

  • Compare INTS10 genomic distribution across different cell types or differentiation states

  • Investigate whether INTS10 recruitment is influenced by specific transcription factors

How can INTS10 antibodies be used to investigate the role of Integrator complex dysregulation in cancer progression?

The Integrator complex, including INTS10, has been implicated in cancer biology. To investigate its role:

• Expression analysis in cancer tissues:

  • Perform immunohistochemistry using INTS10 antibodies on cancer tissue microarrays

  • Compare INTS10 expression between tumor and adjacent normal tissues

  • Correlate expression levels with clinical parameters and patient outcomes, as has been initiated for hepatocellular carcinoma using TCGA data

• Functional studies in cancer cell models:

  • Use INTS10 knockdown/knockout approaches in cancer cell lines to assess effects on:

    • Proliferation and cell cycle progression

    • Migration and invasion capabilities

    • Anchorage-independent growth

  • Perform rescue experiments with wild-type versus mutant INTS10 to identify critical functional domains

• Mechanisms of transcriptional dysregulation:

  • Investigate whether INTS10 modulates oncogene expression through enhancer regulation

  • Assess changes in transcription termination at cancer-relevant genes following INTS10 manipulation

  • Explore potential cancer-specific interaction partners through differential interactome analysis

What experimental approaches can be used to study the dynamics of INTS10 recruitment to chromatin during transcriptional activation?

To investigate the dynamics of INTS10 recruitment during transcriptional activation:

• Inducible transcription systems:

  • Use EGF-responsive gene models, which have shown INTS10 recruitment alongside RNA Polymerase II and INTS11

  • Perform time-course ChIP experiments following transcriptional induction

  • Apply ChIP-seq to map genome-wide recruitment patterns at different time points

• Live-cell imaging approaches:

  • Generate fluorescently tagged INTS10 for real-time visualization

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess binding dynamics

  • Consider optogenetic approaches to induce transcription at specific loci while monitoring INTS10 recruitment

• Sequential ChIP (Re-ChIP):

  • Perform sequential immunoprecipitation to determine co-occupancy of INTS10 with other factors

  • Assess whether INTS10 recruitment precedes or follows RNAPII or other Integrator subunits

  • Combine with nascent RNA analysis to correlate INTS10 binding with transcriptional output