INTS5 Antibody

What is INTS5 and what biological processes is it involved in?

INTS5 (Integrator complex subunit 5) is a critical component of the Integrator complex that associates with the C-terminal domain of RNA polymerase II large subunit. This complex plays a crucial role in the transcription and 3'-box-dependent processing of small nuclear RNAs (snRNAs), particularly U1 and U2 snRNAs . Research has demonstrated that INTS5 functions in hematopoiesis by modulating Smad/BMP signaling pathways . Knockdown studies in zebrafish embryos have shown that INTS5 deficiency affects U1 and U2 snRNA processing, resulting in aberrant splicing of smad1 and smad5 RNA, and reduced expression of hematopoietic genes including stem cell leukemia (scl/tal1) and gata1 . The protein has a molecular weight of approximately 108 kDa and is also known by alternative names INT5 and KIAA1698 .

What are the key molecular characteristics of the INTS5 protein?

INTS5 is characterized by:

• Calculated molecular weight: 108 kDa (confirmed by observed experimental data)

• GenBank Accession Number: BC060841

• Gene ID (NCBI): 80789

• UniProt ID: Q6P9B9

• High sequence conservation across species (e.g., 99% sequence identity between human and mouse/rat for certain epitopes)

The protein functions as part of the multi-subunit Integrator complex, with studies demonstrating that targeting other Integrator subunits leads to similar defects in smad5 RNA splicing and arrested hematopoiesis, suggesting that these proteins function collectively to regulate the BMP pathway during blood cell development .

What types of INTS5 antibodies are available for research applications?

Several types of INTS5 antibodies are available for research:

Most commonly used antibodies are derived from rabbit hosts, targeting specific epitopes of human INTS5. For instance, the immunogen sequence for one commercial antibody is: "DLMGQLSSTYSGQHQRVPHATGALNELLQLWMGCRATRTLMDIYVQCLSALIGSCPDACVDALLDTSVQHSPHFDWVVAHIGSSFPGTII" . Researchers should select antibodies based on their specific experimental requirements and the species being studied.

What applications are INTS5 antibodies validated for?

INTS5 antibodies have been validated for multiple research applications:

It is recommended to optimize antibody concentrations for each specific experimental system to obtain optimal results, as performance can be sample-dependent .

How can INTS5 antibodies be used to study Integrator complex formation and function?

For investigating Integrator complex formation and function:

• Co-immunoprecipitation (Co-IP): Use INTS5 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to pull down the entire Integrator complex from cellular lysates (e.g., HeLa cells) . This allows identification of protein-protein interactions within the complex.

• Chromatin Immunoprecipitation (ChIP): Apply INTS5 antibodies to investigate binding of the Integrator complex to U1 and U2 snRNA genes and identify recruitment sites on chromatin.

• Proximity Ligation Assay (PLA): Combine INTS5 antibodies with antibodies against other Integrator subunits to visualize protein interactions in situ.

• CRISPR-Based Studies: Use INTS5 antibodies for validation of knockout efficiency when creating INTS5-deficient cell lines to study resultant phenotypes.

• Mass Spectrometry Analysis: Following IP with INTS5 antibodies, conduct mass spectrometry to comprehensively identify all interacting partners and post-translational modifications.

Research by Tao et al. demonstrated that targeting different Integrator subunits produces similar phenotypes in zebrafish hematopoiesis, suggesting coordinated function as a complex .

How can INTS5 antibodies be used to investigate RNA processing mechanisms?

To investigate INTS5's role in RNA processing:

• RNA Immunoprecipitation (RIP): Use INTS5 antibodies to capture RNA-protein complexes, followed by RNA isolation and sequencing to identify bound RNAs.

• Nascent RNA Analysis: Combine INTS5 antibody-based techniques with nascent RNA labeling to study the temporal dynamics of snRNA processing.

• iCLIP (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation): Apply INTS5 antibodies to map RNA-protein interaction sites at nucleotide resolution.

• Splicing Analysis: After INTS5 knockdown or knockout, use RT-PCR and RNA-seq in combination with INTS5 antibody validation to examine alterations in splicing patterns of target genes like smad1 and smad5 .

• Immunofluorescence-FISH: Combine INTS5 immunofluorescence (1:20-1:200 dilution) with RNA FISH to visualize co-localization of INTS5 with specific RNA species.

Zebrafish studies have shown that INTS5 knockdown affects U1 and U2 snRNA processing, resulting in aberrant splicing of smad1 and smad5 RNA, establishing a direct link between INTS5 function and RNA processing .

What are the optimal conditions for storing and handling INTS5 antibodies?

Storage and handling recommendations for INTS5 antibodies:

For conjugation-ready formats (e.g., 83154-5-PBS), aliquoting into individual single-use tubes after thawing is recommended to prevent degradation . Always follow manufacturer-specific recommendations as storage conditions can vary between products.

What are the recommended protocols for validating INTS5 antibody specificity?

To ensure antibody specificity:

• Positive and Negative Controls:

  • Use validated positive controls (e.g., HeLa cells, human brain tissue for WB)

  • Include knockout/knockdown samples as negative controls

• Multiple Detection Methods:

  • Cross-validate results using different techniques (WB, IHC, IF)

  • Verify band size matches expected molecular weight (108 kDa)

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be blocked if antibody is specific

• Dilution Series:

  • Test antibody at multiple dilutions (e.g., 1:500-1:6000 for WB)

  • Determine optimal signal-to-noise ratio

• Cross-species Validation:

  • If claiming cross-reactivity, validate in multiple species (human, mouse, rat)

  • Consider sequence homology (e.g., 99% sequence identity between human and mouse/rat for specific epitopes)

• Orthogonal Antibodies:

  • Compare results with antibodies targeting different epitopes of INTS5

  • Consistent results across antibodies support specificity

Western blot protocol recommendations include using SDS-PAGE followed by transfer and probing with INTS5 antibody at dilutions of 1:500-1:5000 for detection in human brain tissue .

What are common issues when using INTS5 antibodies and how can they be resolved?

Common challenges and solutions when working with INTS5 antibodies:

For IHC applications, antigen retrieval is critical - suggested methods include TE buffer pH 9.0 or citrate buffer pH 6.0 .

How should results be interpreted when studying INTS5 in different cell types and experimental conditions?

When interpreting INTS5 experimental results:

• Cell Type Considerations:

  • Expression levels vary across cell types; validated in HeLa, NIH/3T3, SKOV-3 cells

  • Nuclear localization pattern should be verified by IF/ICC (1:20-1:200 dilution)

  • Consider cell-specific interaction partners that may affect antibody accessibility

• Experimental Variables:

  • Stress conditions may alter INTS5 expression or localization

  • Cell cycle phase can impact Integrator complex formation

  • Treatment duration and concentration must be standardized

• Control Interpretation:

  • Always compare to appropriate negative and positive controls

  • Consider using siRNA/shRNA knockdown to validate specificity

• Quantitative Analysis:

  • For Western blots, normalize INTS5 signal to appropriate loading controls

  • For IHC/IF, use standardized scoring systems for expression levels

  • Consider the dynamic range of detection methods

• Cross-species Comparisons:

  • Note the high conservation (99% sequence identity) between human and rodent INTS5 for certain epitopes

  • Species-specific variations in molecular weight or post-translational modifications may occur

• Functional Correlations:

  • In hematopoiesis studies, correlate INTS5 expression with splicing of target RNAs (smad1, smad5) and expression of downstream genes (scl/tal1, gata1)

  • Blood cell differentiation phenotypes should be evaluated alongside molecular data

How has INTS5 antibody research contributed to understanding hematopoiesis and RNA processing?

Research using INTS5 antibodies has significantly advanced our understanding of hematopoiesis and RNA processing:

• Hematopoietic Development:

  • Western blot analysis using INTS5 antibodies demonstrated that morpholino-mediated knockdown of ints5 in zebrafish embryos disrupts protein synthesis during development

  • This knockdown was linked to arrested red blood cell differentiation, similar to scl-deficient embryos

  • INTS5 antibodies helped establish that the protein functions in the Smad/BMP signaling pathway critical for hematopoiesis

• RNA Processing Mechanisms:

  • INTS5 antibodies enabled the demonstration that Integrator complex proteins interact with RNA polymerase II to mediate 3′ end processing of U1 and U2 snRNAs

  • Immunoprecipitation with INTS5 antibodies has helped identify components of the RNA processing machinery that interact with the Integrator complex

• Developmental Biology:

  • INTS5 antibody-based research established a link between RNA processing machinery and downstream effectors of BMP signaling

  • These studies revealed that INTS5 and other Integrator proteins regulate the switch from primitive hematopoietic stem cell identity to blood cell differentiation

This research established INTS5 as part of a previously unidentified regulatory mechanism that connects RNA processing to developmental signaling pathways in blood cell formation.

What emerging applications are being developed for INTS5 antibodies in cancer and developmental biology research?

Emerging applications for INTS5 antibodies include:

• Cancer Research Applications:

  • Detection of INTS5 in prostate cancer tissues using IHC (1:50-1:500 dilution)

  • Analysis of correlation between INTS5 expression and cancer progression

  • Investigation of RNA processing defects in cancer cells using INTS5 as a marker

  • Development of multiplexed detection systems combining INTS5 with other cancer biomarkers

• Developmental Biology:

  • Antibody-based lineage tracing of INTS5-expressing cells during embryonic development

  • Time-course analysis of INTS5 expression during organogenesis

  • Correlation of INTS5 expression with specific developmental transitions

• Stem Cell Research:

  • Monitoring INTS5 during differentiation of hematopoietic stem cells

  • Comparing INTS5 dynamics across different stem cell populations

  • Using INTS5 antibodies to isolate specific progenitor populations

• Multi-omics Integration:

  • Combining INTS5 antibody-based proteomics with transcriptomics and epigenomics

  • Development of antibody-based spatial transcriptomics methods to map INTS5 activity

  • Using INTS5 antibodies in conjunction with nascent RNA sequencing to create integrated maps of transcription and RNA processing

• Therapeutic Relevance:

  • Exploring INTS5 as a potential marker for developmental disorders

  • Investigating the role of INTS5 in cellular responses to RNA-targeted therapeutics

  • Developing screening methods using INTS5 antibodies to identify compounds that modulate RNA processing

As INTS5 research expands, the availability of well-characterized recombinant antibody formats (e.g., 83154-5-PBS) and matched antibody pairs for cytometric bead arrays will facilitate more sophisticated applications in both basic and translational research.