INTS10 Antibody, Biotin conjugated

What is INTS10 and what is its primary function in cellular processes?

INTS10 (Integrator complex subunit 10) is an essential component of the Integrator complex with a calculated molecular weight of 82 kDa, though it is often observed at 70-82 kDa in experimental conditions . It functions primarily as part of the Integrator Arm module along with INTS13, INTS14, and INTS15. This module plays a crucial role in tethering to the core of the Integrator complex . Functionally, INTS10 participates in RNA processing pathways, particularly in the 3' end processing of small nuclear RNAs (snRNAs) and regulation of transcription. The protein's helical repeat structure facilitates its interaction with other Integrator subunits, enabling the complex to perform its regulatory functions in gene expression and RNA processing mechanisms.

How do biotin-conjugated antibodies differ from unconjugated antibodies in research applications?

Biotin-conjugated antibodies contain a covalently attached biotin molecule that enables high-affinity binding to streptavidin or avidin. This conjugation provides significant advantages in detection sensitivity and versatility compared to unconjugated antibodies. In research applications, biotin-conjugated antibodies facilitate signal amplification through the biotin-streptavidin system, which can bind multiple enzyme-conjugated streptavidin molecules to each biotin moiety, thereby enhancing detection sensitivity . Additionally, biotin conjugation enables multiple detection methods using the same primary antibody, as researchers can use various streptavidin-conjugated reporter molecules (fluorophores, enzymes, etc.) without needing different secondary antibodies. Unlike unconjugated antibodies that require a species-specific secondary antibody, biotin-conjugated antibodies can be directly detected, which reduces background and cross-reactivity issues in multi-labeling experiments.

What structural characteristics make INTS10 important for Integrator complex function?

INTS10 possesses a distinctive helical repeat structure that facilitates critical protein-protein interactions within the Integrator complex. The N-terminal helical repeat of INTS10 (residues 1-37) interacts with the C-terminal helices of INTS15 in a head-to-tail arrangement, forming an L-shaped structure that has been confirmed through negative-stain electron microscopy and crosslinking mass spectrometry . This structural arrangement is essential for complex stability, as mutation or deletion of these regions results in nearly complete loss of INTS15 co-purification. Additionally, INTS10's C-terminal region contains residues (including E633 and E634) that are crucial for interaction with INTS14, another key component of the Arm module . These structural features enable INTS10 to serve as a bridging element between different parts of the Integrator complex, facilitating the assembly of higher-order structures necessary for RNA processing and transcriptional regulation.

What are the optimal applications for INTS10 antibodies in molecular biology research?

Based on experimental validation, INTS10 antibodies are optimally suited for Western Blot (WB), Immunoprecipitation (IP), and ELISA applications . For Western Blot analysis, INTS10 antibodies have been successfully tested at dilutions of 1:1000-1:3000, showing distinct bands at 70-82 kDa in multiple cell lines including MCF-7 cells and tissues from various species (human, mouse, rat) . For Immunoprecipitation, INTS10 antibodies perform effectively at concentrations of 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate, particularly in HeLa cells . These applications enable researchers to investigate INTS10's expression levels, post-translational modifications, and protein-protein interactions. For studying the Integrator complex's composition and dynamics, IP using INTS10 antibodies has proven particularly valuable, allowing co-precipitation of interaction partners like INTS13, INTS14, and INTS15 . Additionally, these antibodies can be employed in chromatin immunoprecipitation (ChIP) experiments to investigate INTS10's association with chromatin and its role in transcriptional regulation.

How can researchers validate the specificity of INTS10 antibodies for experimental purposes?

Validating INTS10 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. Researchers should first perform Western blot analysis across multiple cell lines known to express INTS10 (such as HEK-293, HeLa, MCF-7) to confirm detection of the correct molecular weight band (70-82 kDa) . Crucial validation comes from CRISPR-Cas9 knockout or siRNA knockdown experiments, where disappearance of the INTS10 band confirms specificity . For immunoprecipitation experiments, researchers should compare INTS10 antibody pulldowns with IgG control pulldowns, followed by mass spectrometry identification of precipitated proteins to confirm enrichment of known INTS10 interactors (INTS13, INTS14, INTS15) . Additional validation can include using multiple antibodies targeting different epitopes of INTS10 and confirming consistent results. For biotin-conjugated INTS10 antibodies specifically, researchers should compare detection sensitivity and specificity with unconjugated versions in parallel experiments, ensuring the biotin conjugation hasn't altered antibody performance or epitope recognition.

What are the recommended storage and handling conditions for maintaining INTS10 antibody activity?

For optimal preservation of INTS10 antibody activity, storage at -20°C is recommended, where the antibody remains stable for approximately one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity and prevent microbial contamination . Notably, aliquoting is not necessary for -20°C storage of INTS10 antibodies, which simplifies laboratory management. When handling these antibodies, it's essential to avoid repeated freeze-thaw cycles that can lead to protein denaturation and loss of binding efficiency. Before use, allow the antibody to equilibrate to room temperature and gently mix (do not vortex) to ensure homogeneity. For dilution purposes, use buffers appropriate to the specific application (e.g., TBST with 5% non-fat dry milk for Western blotting). Small volume formats (20 μl) may contain 0.1% BSA as a stabilizer , which should be considered when designing experiments sensitive to BSA presence.

How does INTS10 interact with other components of the Integrator complex?

INTS10 engages in two critical interfaces that anchor it within the Integrator complex architecture. First, its N-terminal helical repeat (residues 1-37) forms a head-to-tail interaction with the C-terminal helices of INTS15, resulting in an L-shaped structure essential for complex integrity . Mutation experiments demonstrate that disruption of this interface through W28P/L29P mutations in INTS10 or L384A/L387A mutations in INTS15 abolishes this interaction . Second, INTS10's C-terminal region engages with INTS14, with residues E633 and E634 being particularly critical; mutation of these residues (E633A/E634A) disrupts INTS14 recruitment while leaving INTS15 binding intact . Importantly, INTS10 selectively binds INTS14 but not the structurally similar INTS13, demonstrating binding specificity . Size exclusion chromatography analysis has confirmed that INTS10/15 forms a quaternary complex with INTS5/8, and mixing INTS13/14/10/15 with INTS5/8 results in a high molecular weight peak containing all six proteins . These interactions position INTS10 as a crucial bridging component that helps tether the Integrator Arm module (INTS13/14/10/15) to the core of the complex.

What methodological approaches can effectively isolate INTS10-containing complexes from cellular systems?

Effective isolation of INTS10-containing complexes requires strategic immunoprecipitation approaches that preserve native protein interactions. Researchers have successfully employed epitope-tagged INTS10 expressed in HEK-293T cells followed by affinity purification to isolate intact complexes . For studying endogenous complexes, immunoprecipitation using validated INTS10 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) has proven effective, particularly in HeLa cells . CRISPR-Cas9 mediated genetic engineering to introduce epitope tags at endogenous loci facilitates purification of native INTS10-containing complexes at physiological expression levels . For preserving transient or weak interactions, crosslinking approaches like formaldehyde or DSS treatment prior to lysis can be employed. Differential quantitative proteomics of affinity-purified wild-type versus mutant INTS10 (e.g., W28A/L29A or E633A/E634A) provides valuable insights into interaction networks and complex formation . For biochemical characterization, size exclusion chromatography effectively separates INTS10-containing subcomplexes of different molecular weights, especially when combined with SDS-PAGE and Western blotting or mass spectrometry analysis of fractions .

What functional consequences result from disrupting INTS10's interaction with INTS15 or INTS13/14?

Disruption of INTS10's protein-protein interactions has distinct and significant functional consequences for Integrator complex integrity and function. When the INTS10-INTS15 interaction is disrupted through mutations (e.g., INTS10 W28A/L29A), INTS10 loses its ability to co-precipitate endogenous INTS15 while still retaining INTS13/14 binding capability . This selective disruption demonstrates the modular nature of INTS10's interactions within the complex. More critically, disruption of either interface of INTS10 (with INTS15 or with INTS13/14) affects recruitment of the cleavage module (INTS4/9/11), highlighting how structural integrity of the Arm module is prerequisite for proper assembly of functional Integrator complexes . Additionally, disrupting the INTS14-INTS10 interaction affects recruitment of other proteins such as the uncharacterized ZNF655, suggesting that INTS13/14 may engage additional DNA-binding proteins on chromatin . These findings indicate that INTS10 serves as a critical architectural component that integrates multiple modules of the Integrator complex, and disruption of its interactions can have far-reaching effects on complex assembly, RNA processing, and transcriptional regulation.

How can biotin-conjugated antibodies improve detection sensitivity in INTS10 research?

Biotin-conjugated antibodies significantly enhance detection sensitivity in INTS10 research through the biotin-streptavidin amplification system. The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), allowing for exceptionally stable binding . This property enables signal amplification strategies where multiple streptavidin-conjugated reporter molecules can bind to a single biotin-conjugated antibody, dramatically increasing detection sensitivity. In practical terms, this translates to improved detection of low-abundance INTS10 protein in complex samples or specific subcellular compartments. For immunohistochemistry or immunofluorescence applications, biotin-conjugated antibodies allow for detection of INTS10 in tissues where expression might be limited. Additionally, the small size of biotin (244 Da) minimizes the risk of epitope masking or steric hindrance that might occur with directly-conjugated fluorophores or enzymes. This property is particularly valuable when studying INTS10's interactions within the Integrator complex, where spatial constraints might limit antibody accessibility to certain epitopes.

What are the critical parameters for optimizing INTS10 immunoprecipitation experiments?

Successful INTS10 immunoprecipitation requires careful optimization of several critical parameters to maintain complex integrity while achieving sufficient enrichment. Lysis buffer composition is paramount; a balanced buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, and protease/phosphatase inhibitors provides effective solubilization while preserving protein-protein interactions . The antibody-to-lysate ratio significantly impacts specificity and yield; optimal results for INTS10 IP are achieved using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate . Incubation conditions affect complex stability and antibody binding; overnight incubation at 4°C with gentle rotation provides sufficient time for antibody-antigen interaction while minimizing protein degradation. For INTS10 complex studies, pre-clearing lysates with protein A/G beads reduces non-specific binding. The choice between denaturing and non-denaturing elution depends on experimental goals; non-denaturing methods (competitive elution with excess peptide) preserve complex integrity for downstream functional assays, while denaturing conditions (SDS buffer, boiling) maximize yield for detection purposes. When studying specific INTS10 interactions (e.g., with INTS13/14/15), crosslinking reagents like BS3 or DSP can stabilize transient or weak associations prior to immunoprecipitation .

What considerations should be taken into account when using INTS10 antibodies for chromatin immunoprecipitation (ChIP)?

When performing chromatin immunoprecipitation with INTS10 antibodies, researchers must address several specific considerations to obtain reliable results. First, fixation conditions significantly impact chromatin accessibility and epitope preservation; for INTS10 ChIP, a moderate formaldehyde concentration (1%) with brief fixation time (10 minutes) generally preserves both DNA-protein and protein-protein interactions without causing excessive crosslinking that might mask epitopes . Sonication parameters require optimization to generate DNA fragments of ideal size (200-500 bp) while preserving INTS10-containing complexes. Given INTS10's role in the Integrator complex, using chromatin preparations enriched for transcriptionally active regions (e.g., through nuclear fractionation) may improve signal-to-noise ratio. Antibody validation is crucial; antibodies should be tested for their ability to immunoprecipitate INTS10 from both crosslinked and non-crosslinked samples before proceeding to ChIP experiments. Including appropriate controls is essential: IgG negative controls, input normalization, and positive controls targeting known INTS10-associated genomic regions (e.g., active snRNA genes or regions identified in published INTS10 ChIP-seq datasets). For biotin-conjugated INTS10 antibodies specifically, researchers should use streptavidin-conjugated beads instead of protein A/G beads, and include additional blocking steps with biotin-free BSA to minimize non-specific binding to endogenous biotinylated proteins.

How should researchers interpret discrepancies in INTS10 molecular weight between predicted and observed values?

Discrepancies between INTS10's calculated molecular weight (82 kDa) and its observed molecular weight on Western blots (70-82 kDa) can arise from several biological and technical factors that researchers should systematically evaluate. Post-translational modifications significantly impact migration patterns; phosphorylation can add negative charges that increase apparent molecular weight, while proteolytic processing may reduce it. INTS10 may exist in multiple isoforms resulting from alternative splicing, with the human INTS10 gene potentially encoding variants of different sizes. Protein structure affects SDS binding and migration; INTS10's helical repeat structure may bind SDS differentially compared to globular standard proteins, resulting in anomalous migration. Technical factors also contribute to observed discrepancies: gel percentage, buffer composition, running conditions, and molecular weight marker accuracy can all influence apparent molecular weight measurements. To systematically address these discrepancies, researchers should perform mass spectrometry analysis to determine actual molecular weight, use isoform-specific primers in RT-PCR to identify alternative transcript expression, employ phosphatase treatment to assess contribution of phosphorylation, and compare migration patterns across different gel systems. Notably, consistent observation of INTS10 at 70-82 kDa across multiple studies suggests this is its true migration pattern rather than an experimental artifact .

What are common pitfalls in co-immunoprecipitation experiments involving INTS10 and how can they be addressed?

Co-immunoprecipitation experiments with INTS10 present several potential pitfalls that researchers should proactively address. Insufficient complex solubilization frequently occurs when lysis conditions are too harsh (disrupting protein-protein interactions) or too mild (failing to release complexes from cellular structures); optimize by testing different detergents (NP-40, Triton X-100) at various concentrations . Non-specific binding can generate false positives; address this by including appropriate negative controls (IgG, knockout/knockdown samples), pre-clearing lysates, and using stringent washing conditions calibrated to maintain specific interactions. Antibody cross-reactivity may occur, particularly with structurally similar proteins; validate specificity through knockout/knockdown experiments and consider using multiple antibodies targeting different INTS10 epitopes. Transient or weak interactions may be missed; stabilize these interactions using chemical crosslinkers like DSP or BS3 prior to lysis . Complex stoichiometry variation across cell types and conditions can affect results; address by standardizing experimental conditions and comparing relative rather than absolute interaction quantities. For biotin-conjugated antibodies specifically, endogenous biotinylated proteins may cause background; use avidin pre-clearing of lysates and include biotin-blocking steps to minimize this interference.

How can researchers differentiate between direct and indirect interactions of INTS10 with other proteins?

Differentiating between direct and indirect INTS10 protein interactions requires complementary approaches that progressively eliminate intermediary proteins. In vitro reconstitution with purified recombinant proteins represents the gold standard for establishing direct interactions; researchers have successfully demonstrated direct interactions between INTS10/15 and INTS5/8 using size exclusion chromatography with purified components . Yeast two-hybrid (Y2H) or mammalian two-hybrid assays can identify potential direct interactions in cellular contexts with minimal bridging proteins. For more complex scenarios, protein proximity labeling methods like BioID or APEX2 fused to INTS10 can identify proteins in close physical proximity. Site-directed mutagenesis targeting specific residues at predicted interfaces provides strong evidence for direct interactions when mutations abolish binding; mutations in INTS10 (W28P/L29P affecting INTS15 binding, or E633A/E634A affecting INTS14 binding) have confirmed direct interaction interfaces . Crosslinking mass spectrometry (XL-MS) identifies amino acids in close proximity between proteins, strongly suggesting direct contacts; this approach has validated the INTS10-INTS15 interaction model . Structural approaches including cryo-EM and X-ray crystallography provide definitive evidence of direct interactions when available. Researchers should apply multiple complementary methods, as each has limitations and strengths in distinguishing direct from indirect interactions.

How is INTS10 implicated in disease mechanisms and potential therapeutic targets?

While direct connections between INTS10 and disease mechanisms are still emerging, its critical role in the Integrator complex suggests significant implications for pathological processes. The Integrator complex, including INTS10, regulates the processing of diverse RNA species and transcription termination, processes frequently dysregulated in cancer and neurodevelopmental disorders . INTS10's interaction with the cleavage module (INTS4/9/11) positions it as a potential regulator of 3' end RNA processing, a mechanism implicated in oncogenic alternative polyadenylation . Disruption of INTS10-containing complexes affects recruitment of DNA-binding proteins like ZNF655 to chromatin, potentially altering gene expression patterns relevant to disease progression . INTS10, as part of the Integrator Arm module, participates in binding nucleic acids, suggesting roles in genomic stability maintenance . In therapeutic contexts, targeting protein-protein interactions between INTS10 and other Integrator components (particularly the well-characterized interfaces with INTS14 and INTS15) might offer selective approaches to modulate specific RNA processing events . Developing small molecules or peptide mimetics that specifically disrupt these interfaces without affecting other Integrator functions could provide targeted therapeutic strategies for diseases involving RNA processing dysregulation.

What novel techniques are advancing our understanding of INTS10's dynamic interactions within the cell?

Cutting-edge techniques are revolutionizing our understanding of INTS10's dynamic interactions and functions. CRISPR-Cas9 mediated genetic engineering for endogenous tagging has enabled purification of native Integrator complexes from mammalian cells, providing insights into physiologically relevant INTS10 interactions . Live-cell imaging approaches using fluorescently tagged INTS10 are revealing its spatiotemporal dynamics during transcription and RNA processing. Proximity-dependent biotinylation (BioID, TurboID) fused to INTS10 is mapping its dynamic interactome across different cellular conditions, capturing both stable and transient interactions. AlphaFold2 and other AI-based structural prediction tools have successfully modeled INTS10 structure and its interfaces with partners like INTS15, with predictions validated by experimental approaches . Integrative structural biology combining cryo-EM, crosslinking mass spectrometry, and computational modeling is elucidating the architecture of INTS10-containing complexes . Single-molecule techniques including FRET and super-resolution microscopy are providing insights into INTS10 complex assembly and disassembly kinetics. Global approaches like ChIP-seq and PRO-seq combined with INTS10 perturbation are mapping its genomic occupancy and effects on transcription. These technological advances collectively promise to reveal INTS10's dynamic roles in integrating transcriptional regulation with RNA processing.