INTS13 Antibody

What is INTS13 and what are its primary cellular functions?

INTS13, also known as ASUN (Asunder), is a key component of the Integrator complex involved in various cellular processes. It plays essential roles in maintaining genomic stability and promoting proper gene transcription . INTS13 functions primarily in regulation of the mitotic cell cycle, acting upstream of or within centrosome localization, mitotic spindle organization, and protein localization to the nuclear envelope . This 80 kDa protein is located in both the cytoplasm and nuclear bodies, highlighting its diverse cellular functions .

Research has demonstrated that INTS13 is particularly important in DNA repair mechanisms, making it a significant target for studies in genomic integrity maintenance. Additionally, INTS13 has been identified as a critical factor in developmental processes, with mutations linked to developmental ciliopathies .

How does INTS13 interact with other components of the Integrator complex?

This observation suggests that INTS13 likely recognizes and binds to structural domains formed at the interface between multiple Integrator subunits, rather than binding directly to individual components. Similar interaction patterns have been documented for the INTS4/9/11 Cleavage Module . This complex interaction network is critical for understanding the functional role of INTS13 within the larger Integrator complex and may have implications for developing more specific antibodies targeting different conformational states of INTS13.

What are the key synonyms and identifiers for INTS13 that researchers should know?

When conducting literature searches or database queries related to INTS13, researchers should be aware of several synonyms that have been used to describe this protein:

• ASUN (the most common alternative name)

• GCT1

• NET48

• Mat89Bb

• SPATA30

• C12orf11

In genetic databases, INTS13 is identified by Gene ID 55726 in humans . When working with model organisms, be aware that INTS13 homologs exist across species with varying degrees of conservation. For example, in Ailuropoda melanoleuca (giant panda), INTS13 is identified by Entrez Gene ID 100477387 . The calculated molecular weight of human INTS13 is approximately 80 kDa, which matches the observed molecular weight in western blot analyses .

What criteria should be considered when selecting an INTS13 antibody for research applications?

When selecting an INTS13 antibody for research, several critical factors should be considered to ensure experimental success:

First, evaluate the antibody's specificity through validation data. High-quality INTS13 antibodies should demonstrate specific binding to the target protein with minimal cross-reactivity to other proteins. Review western blot data showing a single band at the expected molecular weight (approximately 80 kDa for full-length INTS13) .

Second, consider the epitope targeted by the antibody. Different regions of INTS13 may be more accessible depending on experimental conditions and protein conformation. Custom antibodies have been raised against distinct regions of INTS13, including the central portion (α-M) and C-terminal region (α-C) . For detecting truncated forms of INTS13, antibodies targeting the central portion may be more effective than those targeting the C-terminus .

Third, select antibodies validated for your specific application. INTS13 antibodies may perform differently in various applications such as western blotting, immunofluorescence, flow cytometry, or immunoprecipitation . Finally, consider the host species to avoid potential cross-reactivity in multi-color immunostaining experiments.

How can researchers validate the specificity of an INTS13 antibody?

Validating antibody specificity is crucial for generating reliable experimental results. For INTS13 antibodies, several validation approaches are recommended:

The most definitive validation method involves using cells with INTS13 knockdown or knockout as negative controls. Researchers have utilized siRNA or shRNA targeting INTS13 to reduce its expression, providing an excellent control for antibody specificity . When examining lysates from control versus INTS13-depleted cells via western blot, a specific INTS13 antibody should show significantly reduced or absent signal in the depleted samples.

Another validation approach utilizes cells from patients with INTS13 mutations. For example, fibroblasts from patients with truncating INTS13 mutations showed no detection with C-terminal antibodies but retained reactivity with antibodies targeting the central region, confirming epitope-specific detection .

For advanced validation, peptide competition assays can be performed, where pre-incubation of the antibody with the immunizing peptide should block specific binding. Additionally, immunoprecipitation followed by mass spectrometry can confirm that the antibody is pulling down authentic INTS13 protein .

What are the typical applications for INTS13 antibodies in research?

INTS13 antibodies have proven valuable across multiple research applications:

In western blotting, INTS13 antibodies detect the protein at approximately 80 kDa and can be used to quantify expression levels across different cell types or experimental conditions . This application is particularly useful for studying the effects of genetic manipulations or drug treatments on INTS13 expression.

Immunofluorescence microscopy utilizing INTS13 antibodies allows visualization of the protein's subcellular localization, revealing its presence in both cytoplasmic and nuclear compartments . This application has been instrumental in understanding INTS13's roles in centrosome localization and nuclear envelope dynamics.

Co-immunoprecipitation experiments with INTS13 antibodies help identify interaction partners within the Integrator complex and other cellular proteins . Such experiments have revealed important interactions with INTS4, INTS9, and INTS11, providing insights into the functional organization of the Integrator complex.

Additionally, INTS13 antibodies have applications in flow cytometry for examining protein expression in heterogeneous cell populations, and in chromatin immunoprecipitation (ChIP) experiments to investigate INTS13's association with specific genomic regions .

What are the optimal conditions for using INTS13 antibodies in western blotting?

For successful western blot detection of INTS13, the following methodological considerations are recommended:

Sample preparation is critical - use fresh cell lysates prepared with RIPA buffer supplemented with protease inhibitors to prevent degradation of INTS13. When working with nuclear proteins, specialized nuclear extraction protocols may improve detection. For INTS13 detection, reducing conditions are required, as demonstrated in validated western blot protocols .

For detection, PVDF membranes often yield better results than nitrocellulose when detecting INTS13. HRP-conjugated secondary antibodies with enhanced chemiluminescence detection provide sufficient sensitivity for endogenous INTS13 detection in most cell types .

How can researchers distinguish between different mutant forms of INTS13 using antibodies?

Distinguishing between wild-type and mutant forms of INTS13 requires strategic selection of antibodies targeting different epitopes:

For truncation mutations, use antibodies targeting different regions of the protein. As demonstrated in patient fibroblast studies, antibodies targeting the C-terminal region (α-C) cannot detect truncated INTS13 proteins that lack this region, while antibodies targeting the central portion (α-M) can still detect these truncated forms . This approach allows researchers to confirm the presence of truncated proteins and estimate their size.

For point mutations affecting protein stability, combine antibody detection with proteasome inhibition. In cells harboring the p.S652L mutation, INTS13 protein levels were significantly reduced due to proteasomal degradation . Treatment with MG132 (proteasome inhibitor) restored protein levels detectable by western blot, confirming that the mutation affects protein stability rather than expression .

For studying nonsense-mediated decay of mutant INTS13 transcripts, combine RT-qPCR with cycloheximide treatment to inhibit NMD, followed by western blotting with appropriate antibodies . This multi-method approach provides comprehensive characterization of how different mutations affect INTS13 at both mRNA and protein levels.

What positive controls should be used when validating INTS13 antibodies?

Proper positive controls are essential for validating INTS13 antibody performance:

Cell lines with confirmed high expression of INTS13 provide excellent positive controls. Based on published research, U266, 293F, and HeLa cells have been validated as positive controls for INTS13 expression . These cell lines consistently show strong, specific bands at the expected 80 kDa molecular weight when probed with validated INTS13 antibodies.

For tissue samples, human placenta and prostate tissues have demonstrated detectable levels of Integrator complex components including INTS13 . When using these tissues as positive controls, proper sample preparation is crucial to preserve protein integrity.

Recombinant INTS13 protein, when available, provides the most definitive positive control. This can be particularly valuable when optimizing new antibodies or troubleshooting detection issues. For advanced applications, overexpression of tagged INTS13 (e.g., FLAG-tagged) in cell lines provides an easily detectable positive control with the added benefit of detection via anti-tag antibodies as a complementary approach .

How can INTS13 antibodies be used to investigate its role in gene expression regulation?

INTS13 antibodies enable sophisticated investigations into its role in transcriptional regulation through several advanced approaches:

Chromatin immunoprecipitation (ChIP) using INTS13 antibodies allows mapping of genomic binding sites, providing insights into which genes may be directly regulated by INTS13. This approach has revealed that INTS13, as part of the Integrator complex, is required for induction of specific genes such as CSF1R during cellular differentiation . ChIP-seq experiments can further expand this to genome-wide binding patterns.

For investigating protein complexes involved in gene regulation, INTS13 antibodies can be used in sequential ChIP (re-ChIP) or co-immunoprecipitation followed by mass spectrometry (IP-MS) to identify co-factors present at specific genomic loci. These techniques have helped establish that INTS13 interacts with NAB2 and other transcriptional regulators .

To study the dynamics of INTS13 recruitment during transcriptional activation, INTS13 antibodies can be employed in chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) at different time points after stimulation. This approach has been instrumental in understanding the temporal relationship between INTS13 recruitment and gene activation during processes such as immediate early gene induction and monocytic differentiation .

What is the relationship between INTS13 mutations and disease pathology, and how can antibodies help study this?

INTS13 mutations have been linked to developmental ciliopathies, and antibodies provide valuable tools for investigating the underlying pathomechanisms:

Antibodies targeting different INTS13 domains can characterize how specific mutations affect protein expression, stability, and localization. Research on patient fibroblasts carrying homozygous INTS13 mutations demonstrated that truncating mutations lead to nonsense-mediated decay of mRNA and production of low levels of truncated protein . Point mutations like p.S652L resulted in unstable proteins targeted for proteasomal degradation . These molecular consequences were elucidated using custom antibodies against different regions of INTS13.

For studying ciliopathy mechanisms, immunofluorescence with INTS13 antibodies can reveal abnormal cellular phenotypes in patient-derived cells. Since INTS13 is involved in centrosome localization and mitotic spindle organization , these antibodies can help visualize defects in these structures that may contribute to ciliopathy pathogenesis.

Tissue-specific expression patterns of INTS13 in affected organs can be examined using immunohistochemistry with validated antibodies. This approach helps correlate INTS13 expression with disease manifestations in different tissues and may reveal tissue-specific roles of INTS13 relevant to understanding the selective vulnerability of certain organs in ciliopathies.

How can INTS13 antibodies contribute to understanding protein complex dynamics within the Integrator complex?

INTS13 antibodies provide sophisticated tools for dissecting the assembly and function of protein complexes:

Co-immunoprecipitation with INTS13 antibodies followed by western blotting for other Integrator subunits can reveal which interactions are stable under different cellular conditions. Research has shown that INTS13 interacts with INTS4 only when INTS9 and INTS11 are simultaneously present, suggesting a complex interaction network within the Integrator complex . These findings highlight the value of INTS13 antibodies in mapping complex protein-protein interactions.

For studying complex dynamics during cellular processes, proximity ligation assays (PLA) using INTS13 antibodies paired with antibodies against other Integrator subunits can visualize transient interactions in situ. This approach provides spatial information about where in the cell these interactions occur, which cannot be obtained from traditional co-immunoprecipitation experiments.

Blue native PAGE combined with INTS13 antibody detection enables analysis of intact native complexes, providing insights into the size and composition of different INTS13-containing complexes. This technique can help determine whether INTS13 exists in sub-complexes or only as part of the complete Integrator complex, contributing to our understanding of Integrator assembly and function .

What are common problems encountered with INTS13 antibodies and how can they be resolved?

Researchers working with INTS13 antibodies may encounter several common challenges:

For weak or absent signals in western blotting, first verify that your cells express detectable levels of INTS13 by using validated positive control cell lines such as U266, 293F, or HeLa cells . If signal remains weak, optimize protein extraction by using specialized buffers for nuclear proteins, as INTS13 is present in nuclear bodies . Increasing antibody concentration or extending incubation time may improve detection. Additionally, ensure fresh protease inhibitors are included in lysis buffers to prevent degradation.

For high background or non-specific bands, optimize blocking conditions by testing different blocking agents (BSA vs. milk) and increasing blocking time. Reducing primary antibody concentration may also improve specificity. For persistent non-specific bands, consider using more stringent washing conditions with higher salt concentrations or longer wash times.

If detecting mutant forms of INTS13 is problematic, choose antibodies targeting appropriate epitopes. For truncating mutations, antibodies targeting regions upstream of the truncation are essential . For unstable mutant proteins, proteasome inhibitors like MG132 may be necessary to prevent degradation before detection .

How can researchers optimize immunoprecipitation protocols for studying INTS13 interactions?

Successful immunoprecipitation of INTS13 requires careful optimization:

The choice of lysis buffer is critical - for studying interactions within the Integrator complex, use mild non-ionic detergents like 0.5% NP-40 or 1% Triton X-100 to preserve protein-protein interactions. For detecting weaker or transient interactions, consider crosslinking cells with formaldehyde before lysis. Nuclear extraction protocols are often necessary since INTS13 is present in nuclear bodies .

For antibody selection, monoclonal antibodies typically provide higher specificity but may recognize limited epitopes. The amount of antibody required should be empirically determined, typically starting with 2-5 μg per immunoprecipitation reaction. Pre-clearing lysates with protein A/G beads before adding the INTS13 antibody can reduce non-specific binding.

To validate immunoprecipitation specificity, always include appropriate controls: an isotype control antibody matched to your INTS13 antibody, and ideally, INTS13-depleted cell lysates as negative controls . For detecting co-immunoprecipitated partners, sequential or reciprocal immunoprecipitation provides the strongest evidence for specific interactions. When studying complex formation with other Integrator subunits, remember that some interactions may only be detected when multiple components are present .

What considerations are important when using INTS13 antibodies in immunofluorescence microscopy?

For optimal immunofluorescence detection of INTS13, consider these methodological refinements:

Permeabilization is critical since INTS13 is found in both cytoplasmic and nuclear compartments . Use 0.1-0.2% Triton X-100 for balanced permeabilization of both compartments. For visualizing nuclear INTS13 specifically, consider stronger permeabilization of the nuclear envelope.

Antibody dilution for immunofluorescence typically requires higher concentrations than western blotting - start with a 1:200 to 1:500 dilution range and optimize. Incubating primary antibodies overnight at 4°C often improves specific signal. Include appropriate controls including secondary-only controls and INTS13-depleted cells when possible.

For co-localization studies with other Integrator components, careful selection of compatible secondary antibodies is essential to avoid cross-reactivity. Sequential staining protocols may be necessary when antibodies are raised in the same host species. When examining INTS13's role in centrosome localization or mitotic spindle organization, co-staining with markers like γ-tubulin can provide valuable contextual information .