INTS6 Antibody, Biotin conjugated

What is INTS6 and why is it important to study?

INTS6 (Integrator complex subunit 6) is a component of the Integrator (INT) complex that plays a critical role in the transcription and 3'-box-dependent processing of small nuclear RNAs (snRNAs) U1 and U2 . The Integrator complex associates with the C-terminal domain of RNA polymerase II largest subunit (POLR2A) and is recruited to U1 and U2 snRNA genes . INTS6 has multiple synonyms in the literature, including Int6, DBI-1, Protein DDX26, Protein deleted in cancer 1 (DICE1), DDX26A, and others . The protein has gained significant research attention because it mediates recruitment of cytoplasmic dynein to the nuclear envelope and, importantly, may function as a tumor suppressor by suppressing tumor cell growth when ectopically expressed . INTS6 also participates in the DNA damage response pathway through direct interactions with other proteins such as INTS3 and hSSB1, as demonstrated through co-immunoprecipitation studies . These multifaceted functions make INTS6 a critical protein to study for understanding both normal cellular processes and disease mechanisms, particularly in cancer biology where tumor suppressor functions are essential for developing potential therapeutic strategies.

What are the key specifications of commercially available INTS6 antibodies?

Commercially available INTS6 antibodies, particularly the biotin-conjugated versions, share several important specifications that researchers should consider when designing experiments. These antibodies are typically rabbit polyclonal antibodies with IgG isotype that specifically target human INTS6 protein . The immunogen used for generating these antibodies varies between manufacturers, with some using a recombinant human Integrator complex subunit 6 protein fragment (amino acids 571-795) while others use synthetic peptides within human INTS6 (amino acids 750-800) . The purification process commonly involves Protein G affinity chromatography, resulting in highly purified antibodies with purity levels typically exceeding 95% . Most INTS6 antibodies are supplied in liquid form with specific buffer compositions (such as 0.01 M PBS, pH 7.4, with preservatives like Proclin-300 and stabilizers like 50% glycerol) . The UniProt ID associated with the target protein is Q9UL03, which allows researchers to cross-reference detailed protein information . The most common application validated for biotin-conjugated INTS6 antibodies is ELISA, with recommended dilutions ranging from 1:500 to 1:1000, though optimal concentrations should be determined empirically by researchers for their specific experimental conditions .

How should INTS6 antibody be stored and handled to maintain its activity?

Proper storage and handling of INTS6 antibody, particularly biotin-conjugated versions, is critical to maintaining its reactivity and ensuring experimental reproducibility over time. The antibody should be stored at -20°C in aliquoted portions to minimize freeze-thaw cycles, which can progressively degrade antibody quality and diminish binding efficacy . When preparing aliquots, researchers should use sterile conditions and appropriate low-protein-binding tubes to prevent contamination and minimize antibody loss through adsorption to container surfaces. The storage buffer typically contains 50% glycerol, which prevents freezing at -20°C and maintains antibody stability over extended periods . It is particularly important to protect biotin-conjugated antibodies from exposure to light, as photobleaching can significantly reduce the detection sensitivity in subsequent applications . Prior to use, the antibody should be thawed gently at 4°C or on ice rather than at room temperature to preserve protein integrity. Vortexing should be avoided as it can cause protein denaturation; instead, gentle inversion or flicking of the tube is recommended for mixing. For long-term storage exceeding six months, some manufacturers recommend keeping the antibody at -80°C, though this may vary based on specific formulations. Researchers should always document the number of freeze-thaw cycles and monitor antibody performance over time, particularly if using older stocks for critical experiments where sensitivity and specificity are paramount.

Which applications have been validated for INTS6 antibody, biotin conjugated?

The biotin-conjugated INTS6 antibody has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications with recommended dilutions typically ranging from 1:500 to 1:1000 . While ELISA represents the most thoroughly validated application, the underlying characteristics of this antibody suggest potential utility in other immunodetection methods that leverage biotin-streptavidin interactions. The antibody's high purity (>95%) and specific targeting of human INTS6 make it potentially suitable for immunoprecipitation experiments when coupled with streptavidin-based capture systems, though researchers should conduct preliminary optimization studies . Unlike non-conjugated INTS6 antibodies that have been validated for Western blotting and immunohistochemistry on formalin-fixed paraffin-embedded tissues (IHC-P), the biotin-conjugated version may require additional validation for these applications . When considering immunohistochemistry applications, researchers should note that the specificity of biotinylation methods significantly impacts results, with protein A Z-domain (ZBPA) biotinylation showing superior specificity compared to non-specific conjugation methods like Lightning-Link, which can result in off-target staining patterns . Researchers pursuing novel applications should conduct thorough positive and negative controls, potentially including knockdown validation using INTS6 siRNA to confirm specificity, similar to approaches used in developing antibodies against endogenous INTS6 .

How does biotin conjugation affect INTS6 antibody specificity and performance?

The method of biotin conjugation substantially impacts INTS6 antibody specificity and performance in downstream applications, particularly for tissue-based detection systems. Research comparing different biotinylation methods has demonstrated that techniques specifically targeting the Fc portion of antibodies, such as the modified Z-domain of protein A (ZBPA) method, produce significantly more specific staining patterns compared to non-directed conjugation approaches . When non-specific conjugation methods like Lightning-Link are used, they can biotinylate not only the antibody of interest but also stabilizing proteins commonly present in antibody preparations (such as albumin or gelatin), resulting in characteristic off-target background staining patterns across multiple tissue types . This non-specific staining typically manifests as nuclear positivity in tissues like tonsil and cerebellum, and nuclear/cytoplasmic positivity in uterus, placenta, intestine, cerebral cortex, and pancreas . ZBPA biotinylation, in contrast, provides more stringent immunostaining by preventing conjugation to non-IgG proteins, though it generally requires higher antibody concentrations to achieve comparable signal intensity . The trade-off between specificity and sensitivity creates important considerations for researchers: while non-specific methods may offer greater apparent sensitivity, they risk generating misleading results due to background staining. For critical research applications involving INTS6 detection in tissues or cells, researchers should prioritize conjugation methods that specifically target the antibody's Fc region, validate staining patterns against unconjugated antibody controls, and optimize antibody concentrations to balance specificity and sensitivity for their particular experimental system.

What are the biochemical interactions between INTS6 and other components of the DNA damage response pathway?

INTS6 participates in the DNA damage response pathway through direct interactions with multiple protein partners, forming functional complexes that mediate critical cellular processes. Co-immunoprecipitation (co-IP) and reverse co-IP experiments using specific antibodies against endogenous INTS6 have conclusively demonstrated that INTS6 physically associates with INTS3 and hSSB1 in vivo . Using antibodies that recognize a specific 100 kDa polypeptide (validated through siRNA knockdown experiments), researchers have characterized these protein-protein interactions as part of a core hSSB1-INTS complex that participates in DNA damage response mechanisms . The interaction between INTS6 and hSSB1 is particularly significant as hSSB1 is a single-stranded DNA-binding protein that plays a crucial role in sensing and responding to DNA damage, suggesting that INTS6 may function in early detection and signaling of DNA lesions . Beyond these primary interactions, INTS6 as a component of the Integrator complex associates with the C-terminal domain of RNA polymerase II, linking it to transcriptional regulation and processing of small nuclear RNAs . Additional research suggests that INTS6 mediates recruitment of cytoplasmic dynein to the nuclear envelope, indicating a potential role in nuclear-cytoplasmic transport mechanisms that may be relevant during DNA damage responses . These multifaceted interactions position INTS6 at the intersection of RNA processing, transcriptional regulation, and DNA damage response pathways, highlighting its importance as a multifunctional adaptor protein in maintaining genomic integrity.

How can researchers optimize ELISA protocols using INTS6 antibody, biotin conjugated?

Optimizing ELISA protocols with biotin-conjugated INTS6 antibody requires systematic adjustment of multiple parameters to achieve maximum sensitivity and specificity. While manufacturers recommend dilutions between 1:500 and 1:1000 as starting points, researchers should conduct preliminary titration experiments using serial dilutions (e.g., 1:250, 1:500, 1:1000, 1:2000) to determine the optimal antibody concentration for their specific sample types and detection systems . The coating buffer composition and pH significantly impact antigen adsorption to ELISA plates; for INTS6 detection, researchers should compare carbonate buffer (pH 9.6) with phosphate-buffered saline (PBS, pH 7.4) to determine which provides better sensitivity. Blocking solutions require careful optimization—while 1-5% BSA in PBS with 0.05% Tween-20 is commonly used, researchers should test alternatives like casein or commercial blocking buffers that may reduce background without compromising specific signal. The streptavidin-HRP conjugate concentration must be balanced to provide sufficient signal amplification without increasing non-specific binding; a titration range of 1:1000 to 1:10,000 is recommended for initial testing. Incubation conditions significantly impact assay performance—lengthening primary antibody incubation time (from standard 1-2 hours to overnight at 4°C) often improves sensitivity, particularly for low-abundance targets like INTS6 in complex biological samples. Wash steps should be optimized for both buffer composition (PBS-T vs. TBS-T) and number of washes (typically 3-5 times), with additional washes potentially reducing background in problematic samples. Detection substrate selection between colorimetric (TMB), chemiluminescent, or fluorescent options should be based on required sensitivity, with chemiluminescent substrates typically offering 10-100 fold greater sensitivity for detecting low levels of INTS6 in experimental samples.

What are the methodological considerations for using INTS6 antibody in tumor suppressor research?

Investigating INTS6's tumor suppressor function requires careful methodological considerations across multiple experimental platforms. Since INTS6 has been identified as having ectopic expression that suppresses tumor cell growth, researchers should design experiments that examine both loss and gain of function within appropriate cancer models . Cell line selection is critical—researchers should prioritize cell lines with documented INTS6 expression (or silencing) relevant to their cancer type of interest, validating expression levels via RT-qPCR and Western blotting with well-characterized INTS6 antibodies before proceeding with functional studies. For loss-of-function studies, siRNA or CRISPR-Cas9 approaches targeting INTS6 can be employed, followed by comprehensive phenotypic analysis including proliferation assays (MTT/XTT, BrdU incorporation), colony formation assays, cell cycle analysis (flow cytometry), and apoptosis assessment (Annexin V/PI staining). Gain-of-function studies should utilize controlled expression systems (tetracycline-inducible or lentiviral vectors) to reintroduce INTS6 into deficient cell lines while monitoring the same phenotypic endpoints. Biotin-conjugated INTS6 antibodies can be particularly valuable for chromatin immunoprecipitation (ChIP) experiments when coupled with streptavidin-based capture systems to identify genomic binding sites that might explain INTS6's tumor suppressor activity. For translational relevance, researchers should consider tissue microarray analysis of clinical samples using validated INTS6 antibodies (potentially including biotin-conjugated versions with specific conjugation methods like ZBPA) to correlate expression patterns with patient outcomes, tumor stage, and other clinicopathological parameters . Throughout these experiments, researchers must include appropriate controls for antibody specificity, including INTS6-depleted samples, to ensure that observed effects are specifically attributable to INTS6 function rather than experimental artifacts or off-target effects.

How can researchers validate the specificity of INTS6 antibody in their experimental system?

Validating the specificity of INTS6 antibody, particularly biotin-conjugated versions, requires a multi-faceted approach incorporating both positive and negative controls across several validation techniques. The gold standard validation method involves gene silencing through siRNA targeting INTS6, followed by immunodetection to confirm reduced signal intensity corresponding to protein knockdown, as demonstrated in previous studies developing antibodies against endogenous INTS6 . Western blot analysis should show a single band at the expected molecular weight of approximately 100 kDa, with diminished intensity in knockdown samples; multiple bands or unexpected molecular weights may indicate cross-reactivity or degradation products requiring further optimization . Peptide competition assays provide another validation approach—pre-incubating the INTS6 antibody with excess immunizing peptide (corresponding to amino acids 571-795 or 750-800, depending on the antibody) should substantially reduce or eliminate specific signals in subsequent applications . For tissue or cell-based applications, researchers should compare staining patterns between biotin-conjugated and unconjugated INTS6 antibodies, considering the differential impact of biotinylation methods on specificity . Testing across multiple applications (ELISA, Western blot, immunohistochemistry) with consistent results increases confidence in antibody specificity, while discrepancies may indicate application-specific interferences. When using biotin-conjugated antibodies, additional controls for endogenous biotin interference should be included, particularly for biotin-rich tissues like liver, kidney, and adipose tissue. Finally, orthogonal validation using different antibody clones targeting distinct INTS6 epitopes, or complementary methodologies like mass spectrometry or recombinant expression systems, provides the highest level of confidence in specific INTS6 detection within experimental systems.

What are common pitfalls when working with biotin-conjugated antibodies in immunohistochemistry?

Immunohistochemistry (IHC) with biotin-conjugated antibodies presents several potential pitfalls that researchers should actively address to ensure reliable results. The most significant challenge is endogenous biotin interference, particularly in biotin-rich tissues like liver, kidney, brain, and mammary gland, which can produce false-positive signals independent of target protein expression . This issue can be mitigated by implementing specific blocking steps using commercial avidin/biotin blocking kits or through endogenous biotin blocking with unconjugated streptavidin followed by free biotin saturation before applying the biotin-conjugated primary antibody. The biotinylation method substantially impacts specificity and background staining—non-specific conjugation approaches like Lightning-Link can result in biotinylation of stabilizing proteins (albumin, gelatin) in antibody preparations, generating characteristic non-specific staining patterns across multiple tissues . This biotinylation-related background appears as nuclear positivity in tissues like tonsil and cerebellum, with nuclear/cytoplasmic signals in various other tissues, and cannot be eliminated through simple filtering to remove free biotin . Signal amplification systems using avidin-biotin complexes (ABC) or tyramide signal amplification can enhance sensitivity but may exacerbate background issues; researchers should carefully titrate detection reagents and consider alternative approaches like polymer-based detection systems when background persists. Tissue fixation conditions significantly impact epitope availability—overfixation can mask epitopes while underfixation compromises tissue morphology; optimization of antigen retrieval methods (heat-induced versus enzymatic) is often necessary. The biotin-streptavidin interaction is pH-sensitive, functioning optimally between pH 4-7; researchers should ensure that rinse buffers and dilution media maintain appropriate pH to preserve detection sensitivity across staining protocols.

How should researchers design experiments to study INTS6 interactions with INTS3 and hSSB1?

Designing experiments to investigate INTS6 interactions with INTS3 and hSSB1 requires a comprehensive, multi-technique approach to characterize these protein-protein associations in various cellular contexts. Co-immunoprecipitation (co-IP) experiments represent the foundation of interaction studies and should include both forward (immunoprecipitating INTS6 and probing for INTS3/hSSB1) and reverse approaches (immunoprecipitating INTS3 or hSSB1 and probing for INTS6) to confirm bidirectional associations . These experiments should be performed under both normal conditions and following DNA damage induction (e.g., using ionizing radiation, UV exposure, or chemical agents like etoposide) to assess whether these interactions are constitutive or damage-responsive . Proximity ligation assays (PLA) offer an in situ approach to visualize protein interactions within intact cells, allowing researchers to determine the subcellular localization of INTS6-INTS3-hSSB1 complexes and how this may change following DNA damage. Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) techniques using fluorescently-tagged proteins can provide quantitative measurements of protein proximity in living cells. Domain mapping experiments using truncated protein constructs are essential for identifying the specific regions mediating these interactions; researchers should design a series of deletion and point mutation constructs covering the INTS6 protein (particularly focusing on regions between amino acids 571-795, which have been used as immunogens for antibody development) . For functional characterization, researchers should implement siRNA knockdown of individual components (INTS6, INTS3, or hSSB1) followed by phenotypic analysis of DNA damage responses including γH2AX foci formation, cell cycle checkpoint activation, and repair pathway choice (homologous recombination versus non-homologous end joining). Chromatin immunoprecipitation (ChIP) using biotin-conjugated INTS6 antibodies with streptavidin-based capture can help identify whether these protein complexes associate with specific genomic regions, potentially linking their interactions to transcriptional regulation functions.

What controls should be included when using INTS6 antibody in ELISA experiments?

Comprehensive controls are essential when using biotin-conjugated INTS6 antibody in ELISA experiments to ensure valid and interpretable results. Antigen standard curves using recombinant INTS6 protein (preferably the same region used as immunogen, amino acids 571-795) should be included in each experiment to establish detection limits and quantification parameters . A minimum of three technical replicates should be performed for all samples and standards to assess assay precision and allow statistical analysis of variations. Negative control wells should include: (1) no primary antibody control (buffer only) to assess non-specific binding of detection reagents; (2) isotype control (biotin-conjugated rabbit IgG at the same concentration) to identify potential non-specific binding related to antibody class rather than antigen specificity; and (3) irrelevant target control (biotin-conjugated antibody against an unrelated protein) to evaluate specificity of the detection system . Positive controls are equally important and should include: (1) samples with confirmed INTS6 expression (e.g., cell lysates from lines known to express INTS6); (2) spike-in controls where recombinant INTS6 is added to sample matrix to assess matrix effects and recovery; and (3) dilution linearity tests where samples are serially diluted to confirm that measurements remain proportional to concentration, confirming assay is within dynamic range. Additional validation controls should include pre-absorption controls (antibody pre-incubated with immunizing peptide) to confirm signal specificity, and cross-reactivity assessment using related proteins (other Integrator complex components) to ensure the antibody specifically recognizes INTS6 rather than homologous proteins . For biotin-conjugated antibodies specifically, endogenous biotin controls (streptavidin-HRP only, no primary antibody) help identify potential interference from endogenous biotin in biological samples, which can be particularly important when working with biotin-rich tissue extracts.

How does INTS6 expression correlate with tumor progression in different cancer types?

The relationship between INTS6 expression and tumor progression appears to be complex and potentially cancer type-specific, reflecting its proposed role as a tumor suppressor. Multiple studies have identified INTS6 (also known as DICE1) as having "ectopic expression suppressing tumor cell growth," suggesting that its normal expression may inhibit cancer progression while its downregulation or loss may contribute to tumor development . This tumor suppressor function aligns with INTS6's alternative name "Protein deleted in cancer 1," indicating that genomic loss of this gene has been observed in certain malignancies . While the available search results don't provide specific correlation data across different cancer types, the consistent identification of INTS6 as a potential tumor suppressor suggests that decreased expression would generally correlate with more aggressive disease phenotypes, poorer prognosis, or advanced tumor stages. Mechanistically, INTS6's participation in the DNA damage response pathway through interactions with proteins like INTS3 and hSSB1 provides a potential explanation for its tumor suppressive activity, as compromised DNA damage response can lead to genomic instability—a hallmark of cancer progression . As a component of the Integrator complex involved in snRNA processing and transcriptional regulation, INTS6 downregulation may also disrupt normal RNA processing pathways that influence cancer-related gene expression programs . Researchers investigating INTS6 expression in specific cancer types should consider both protein-level detection using validated antibodies and transcript-level analysis, as well as potential post-translational modifications or protein-protein interactions that might be altered during cancer progression, potentially influencing INTS6's tumor suppressive functions beyond simple expression level correlations.

What methodological approaches can resolve discrepancies in INTS6 detection across different experimental platforms?

Resolving discrepancies in INTS6 detection across different experimental platforms requires systematic troubleshooting and integration of complementary approaches to distinguish technical artifacts from genuine biological variations. When Western blot and ELISA results conflict, researchers should first ensure antibody specificity through validation techniques like siRNA knockdown, which has been successfully used to verify the specificity of antibodies recognizing endogenous INTS6 as a 100 kDa polypeptide . Post-translational modifications may cause platform-specific detection differences; therefore, researchers should consider using phosphatase or deglycosylase treatments of samples prior to analysis to determine if modifications affect epitope accessibility in different applications. For discrepancies between protein-level detection (immunoassays) and transcript-level measurements (RT-qPCR), researchers should examine potential post-transcriptional regulation mechanisms such as miRNA targeting or differential protein stability across tissue types. Sample preparation variables significantly impact detection—different extraction buffers, fixation methods, or epitope retrieval techniques can dramatically alter INTS6 detection sensitivity; standardizing these protocols across platforms helps isolate true biological differences from methodology-induced variations. When immunohistochemistry results differ from other methods, researchers should consider the impact of biotinylation techniques on antibody specificity, as studies have demonstrated that different conjugation methods (ZBPA versus Lightning-Link) produce distinctly different staining patterns, with more targeted biotinylation approaches showing superior specificity . Cross-platform validation using orthogonal methods remains the gold standard; mass spectrometry-based proteomics can provide antibody-independent confirmation of INTS6 presence and potential interacting partners. For particularly challenging discrepancies, epitope mapping studies using peptide arrays or recombinant protein fragments can identify which regions of INTS6 are accessible in different experimental contexts, potentially explaining platform-specific detection limitations.

What analytical approaches can differentiate between specific and non-specific signals when using biotin-conjugated antibodies?

Distinguishing between specific and non-specific signals when using biotin-conjugated antibodies requires rigorous analytical approaches that systematically eliminate potential sources of background. Comparative analysis between staining patterns obtained with biotin-conjugated antibodies and their unconjugated counterparts provides crucial baseline information—the specific signal should show the same localization and relative intensity across both formats, while additional signals appearing only with biotinylated antibodies likely represent artifacts . Research has demonstrated that the biotinylation method significantly impacts specificity; antibodies conjugated using techniques that specifically target the Fc portion (like ZBPA) show distinctly different staining patterns compared to non-specific conjugation methods (like Lightning-Link), which can biotinylate other proteins present in antibody preparations . The characteristic non-specific staining pattern generated by non-specifically biotinylated proteins shows nuclear positivity in tissues like tonsil and cerebellum, and nuclear/cytoplasmic signals in tissues including uterus, placenta, and intestine—researchers should be vigilant for this pattern when analyzing results . Peptide competition assays provide powerful analytical validation—pre-incubating the biotin-conjugated INTS6 antibody with excess immunizing peptide should substantially reduce specific signals while leaving non-specific background unchanged, allowing computational subtraction of background in image analysis. Digital image analysis tools can be employed to quantify signal-to-noise ratios across different tissues or cellular compartments, potentially revealing statistical patterns that distinguish specific from non-specific labeling. Multi-parameter analysis combining the biotin-conjugated INTS6 antibody with differently labeled antibodies against known INTS6-interacting partners (like INTS3 or hSSB1) can reveal biologically relevant co-localization patterns that are unlikely to occur with non-specific signals . For quantitative applications, standard curve analysis using samples with confirmed INTS6 depletion (through siRNA) can establish detection thresholds that minimize false positives from non-specific binding . Ultimately, researchers should implement a weight-of-evidence approach, considering multiple analytical perspectives to confidently distinguish biologically meaningful INTS6 signals from technical artifacts when using biotin-conjugated antibodies.