INTS3 Antibody, FITC conjugated

What is INTS3 and what cellular functions does it participate in?

INTS3, also known as Integrator Complex Subunit 3, functions as a critical component of the Integrator complex that participates in small nuclear RNA (snRNA) processing and RNA polymerase II (RNAPII) pause-release mechanisms. The protein is encoded by the INTS3 gene and has several synonyms including C1orf193, C1orf60, INT3, and sensor of single-strand DNA complex subunit A (SOSSA) . INTS3 plays a fundamental role in transcriptional regulation by facilitating proper processing of snRNAs, which are essential components of the spliceosome machinery .

In addition to its role in RNA processing, INTS3 contributes to genome stability and DNA damage responses through its interaction with NABP (nucleic acid binding protein). This multifunctional nature makes INTS3 a particularly important target for research investigating both normal cellular processes and pathological conditions such as leukemia, where its aberrant splicing has been implicated in disease progression .

What experimental applications are suitable for INTS3 antibody, FITC conjugated?

FITC-conjugated INTS3 antibodies are versatile research tools applicable to multiple experimental techniques:

• Immunofluorescence (IF): Particularly useful for both paraffin-embedded sections and frozen tissues, allowing direct visualization of INTS3 localization without the need for secondary antibodies

• Western Blotting (WB): Enables detection of INTS3 protein expression levels and molecular weight confirmation

• Immunohistochemistry (IHC): Applicable for paraffin-embedded sections (IHC-P) and frozen sections (IHC-Fr) to examine INTS3 distribution in tissue contexts

• Flow Cytometry: The FITC conjugate provides direct fluorescence detection capability for analyzing INTS3 in cell populations

The recommended dilution ranges for immunofluorescence applications are typically between 1:50-1:200, though optimization may be necessary for specific experimental conditions .

What species reactivity can be expected with commercially available INTS3 antibody, FITC conjugated?

The species reactivity profiles of FITC-conjugated INTS3 antibodies can vary by manufacturer and catalog number. Based on the available information:

This broad cross-reactivity, particularly with the ORB4044 antibody, makes these reagents valuable for comparative studies across multiple model organisms . Researchers should verify reactivity in their specific experimental systems, as performance can vary depending on tissue preparation and experimental conditions.

How should researchers validate the specificity of INTS3 antibody, FITC conjugated?

To ensure reliable experimental results, proper validation of INTS3 antibody specificity should include:

• Positive and negative control samples: Include tissues or cell lines known to express or lack INTS3 expression

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• RNA interference validation: Compare staining in cells with INTS3 knockdown versus control cells

• Western blot verification: Confirm detection of a band at the expected molecular weight (~118 kDa for human INTS3)

• Correlation with other detection methods: Compare results with alternative INTS3 antibodies or mRNA expression data

For FITC-conjugated antibodies specifically, an additional control using an isotype-matched FITC-conjugated irrelevant antibody helps rule out non-specific binding from the conjugate itself or the host species immunoglobulin .

What are the recommended storage and handling conditions for INTS3 antibody, FITC conjugated?

Proper storage and handling of FITC-conjugated INTS3 antibodies is crucial for maintaining reagent performance:

• Storage temperature: Store at -20°C for long-term stability

• Light protection: FITC is sensitive to photobleaching, so store in amber vials or wrapped in aluminum foil

• Aliquoting: Divide into small single-use aliquots to avoid repeated freeze-thaw cycles

• Working dilutions: Prepare fresh working dilutions on the day of the experiment

• Buffer composition: Store in buffered solutions containing stabilizers (typically PBS with preservatives)

• Expiration: Follow manufacturer's recommendations for shelf-life (typically 12-24 months from date of receipt)

Additionally, FITC has optimal fluorescence at slightly alkaline pH (7.5-8.5), so buffer conditions during experiments should be maintained accordingly for maximum signal intensity.

How can INTS3 antibody, FITC conjugated be used to investigate aberrant RNA splicing in leukemia research?

INTS3 has emerged as a key player in leukemogenesis, particularly in the context of IDH2 and SRSF2 mutations. FITC-conjugated INTS3 antibodies provide valuable tools for investigating these mechanisms through:

• Co-localization studies: Using FITC-INTS3 antibodies in combination with antibodies against splicing factors (like SRSF2) to visualize their spatial relationships in leukemic cells

• Monitoring INTS3 expression changes: Quantifying INTS3 protein levels in response to treatments targeting epigenetic modifications or splicing

• Tracking cellular distribution: Determining if mutant SRSF2 alters the subcellular localization of INTS3

• Cell sorting applications: Isolating cell populations with different INTS3 expression levels for further analysis

Research has demonstrated that aberrant INTS3 splicing contributes to leukemogenesis in concert with mutant IDH2 and is dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA . Using FITC-conjugated INTS3 antibodies allows direct visualization of these alterations in protein expression resulting from splicing defects.

What methodological approaches can researchers use to study INTS3-mediated RNA polymerase II regulation?

INTS3 plays a significant role in RNAPII pause-release, and FITC-conjugated INTS3 antibodies can help elucidate this function through:

• Chromatin immunoprecipitation followed by immunofluorescence (ChIP-IF): Combining ChIP with immunofluorescence to visualize INTS3 association with specific genomic regions

• Proximity ligation assays: Detecting interactions between INTS3 and RNAPII components using FITC-INTS3 antibody paired with antibodies against RNAPII subunits

• Live cell imaging: Monitoring INTS3 dynamics during transcription elongation in real-time

• FRAP (Fluorescence Recovery After Photobleaching): Studying the kinetics of INTS3 recruitment to transcription sites

Research has shown striking accumulation of RNAPII across the INTS3 locus in IDH2/SRSF2 double-mutant cells, indicating a feedback loop where INTS3 splicing affects RNAPII activity, which in turn impacts INTS3 expression . FITC-conjugated antibodies facilitate visualization of these complex regulatory mechanisms.

How can researchers optimize INTS3 antibody, FITC conjugated for dual immunofluorescence studies?

For effective dual immunofluorescence studies involving FITC-conjugated INTS3 antibodies:

• Spectral considerations:

  • FITC excites at 495nm and emits at 519nm (green spectrum)

  • Pair with fluorophores having minimal spectral overlap (e.g., Cy3, Cy5, or APC)

  • Use appropriate filter sets to minimize bleed-through

• Sequential staining approach:

  • Apply the FITC-conjugated INTS3 antibody first

  • Block any remaining binding sites

  • Apply the second primary antibody (with a different host species if possible)

  • Follow with an appropriate fluorescently-labeled secondary antibody

• Control experiments:

  • Single-stained controls to set compensation

  • Secondary-only controls to assess non-specific binding

  • Isotype controls for both antibodies

• Recommended starting dilution: 1:50-1:200 for paraffin sections, with optimization required for specific tissues

For studying INTS3's interaction with the Integrator complex or splicing machinery, this approach allows simultaneous visualization of multiple components in the same cellular context.

What insights has INTS3 antibody research provided into the relationship between epigenetic regulation and splicing?

Research utilizing INTS3 antibodies has revealed crucial insights into the interplay between epigenetic regulation and RNA splicing:

• DNA methylation effects: Studies have shown that IDH2 mutations promote increased DNA methylation that impacts INTS3 splicing. Genome-wide maps of DNA cytosine methylation revealed that differentially spliced events in IDH2 mutant and IDH2/SRSF2 double-mutant AML harbor significant hypermethylation of DNA .

• Coordination between mutations: INTS3 investigations demonstrated that IDH2 and SRSF2 mutations coordinately dysregulate splicing through alterations in RNAPII stalling in addition to aberrant sequence recognition of cis elements in RNA .

• Therapeutic implications: Treatment with 5-AZA-CdR (a DNA methyltransferase inhibitor) significantly reduced RNAPII stalling and decreased aberrant INTS3 splicing, suggesting potential therapeutic approaches .

FITC-conjugated INTS3 antibodies can help visualize these effects at the protein level, complementing molecular studies of splicing and methylation patterns.

What factors affect the sensitivity and specificity of INTS3 detection in complex tissue samples?

Several factors influence the performance of FITC-conjugated INTS3 antibodies in complex tissue samples:

• Antibody characteristics:

  • Polyclonal antibodies (like those in and ) provide higher sensitivity but potentially lower specificity

  • The binding epitope (e.g., AA 1-50 in ABIN1403030) affects accessibility in fixed tissues

  • KLH conjugated synthetic peptide immunogens may provide different epitope recognition than recombinant protein immunogens

• Tissue preparation factors:

  • Fixation method and duration affects epitope availability

  • Antigen retrieval methods (heat-induced vs. enzymatic)

  • Section thickness (optimal is typically 4-6 μm)

  • Mounting media (use anti-fade formulations to preserve FITC signal)

• Experimental conditions:

  • Blocking protocols to reduce background

  • Antibody concentration and incubation time

  • Buffer composition and pH (FITC fluorescence is pH-sensitive)

  • Washing stringency

Using appropriate controls and optimization steps for each specific tissue type is essential for achieving reliable INTS3 detection.

What troubleshooting steps should be taken when INTS3 antibody, FITC conjugated shows high background or low signal?

When encountering common issues with FITC-conjugated INTS3 antibodies, systematic troubleshooting can improve results:

For high background:

• Increase blocking time and concentration (5% BSA or normal serum from the same species as secondary antibody)

• Reduce primary antibody concentration (try 1:200 instead of 1:50)

• Increase washing duration and number of washes

• Use 0.1-0.3% Triton X-100 in wash buffer to reduce non-specific binding

• Include 0.1-0.3% Tween-20 in antibody diluent

• Pre-absorb antibody with tissue powder if cross-reactivity is suspected

For low signal:

• Optimize antigen retrieval (try heat-induced epitope retrieval at pH 6.0 and pH 9.0)

• Increase antibody concentration (try 1:50 instead of 1:200)

• Extend primary antibody incubation time (overnight at 4°C)

• Use signal amplification systems compatible with FITC

• Ensure FITC has not been subject to photobleaching

• Check pH of buffers (FITC has optimal fluorescence at pH 7.5-8.5)

These approaches should be systematically tested to determine optimal conditions for specific experimental systems.

How can researchers quantify INTS3 expression levels using FITC-conjugated antibodies?

For quantitative analysis of INTS3 expression using FITC-conjugated antibodies:

• Flow cytometry quantification:

  • Use standardized beads with known fluorescence intensity

  • Calculate molecules of equivalent soluble fluorochrome (MESF)

  • Compare median fluorescence intensity (MFI) between samples

  • Include appropriate isotype controls

• Fluorescence microscopy quantification:

  • Capture images using identical exposure settings

  • Measure mean fluorescence intensity in defined regions of interest

  • Use software like ImageJ or CellProfiler for automated analysis

  • Include internal reference standards

• Considerations for accurate quantification:

  • Account for autofluorescence using unstained controls

  • Include positive controls with known INTS3 expression levels

  • Ensure measurements are within the linear range of detection

  • Consider photobleaching effects in time-course experiments

This quantitative approach is particularly valuable when studying alterations in INTS3 expression due to splicing abnormalities in conditions like leukemia .

What control samples are essential when using INTS3 antibody, FITC conjugated in research?

A comprehensive control strategy for FITC-conjugated INTS3 antibody experiments should include:

• Essential negative controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration

  • Peptide-blocked antibody: INTS3 antibody pre-incubated with immunizing peptide

  • INTS3-knockdown cells: Samples with verified INTS3 reduction via siRNA/shRNA

• Essential positive controls:

  • Cell lines with confirmed INTS3 expression (e.g., certain leukemia lines)

  • Tissues known to express INTS3 (based on public database information)

  • Recombinant INTS3 protein samples (for Western blot controls)

• Technical controls:

  • Single-stained samples (for multicolor experiments)

  • Unstained samples (for autofluorescence assessment)

  • Fixation controls (to assess fixation effects on epitope recognition)

These controls help distinguish specific INTS3 staining from technical artifacts and provide crucial validation for experimental findings.

What are the optimal protocols for using INTS3 antibody, FITC conjugated in flow cytometry?

For flow cytometry applications with FITC-conjugated INTS3 antibodies:

• Sample preparation:

  • Harvest cells (1-5 × 10^6 cells per sample)

  • Fix with 2-4% paraformaldehyde (15-20 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 or saponin buffer (10 minutes)

• Staining procedure:

  • Block with 3-5% BSA or normal serum (30 minutes)

  • Incubate with FITC-conjugated INTS3 antibody (1:50-1:100 dilution, 45-60 minutes)

  • Wash 3× with PBS containing 1% BSA

  • Resuspend in appropriate flow cytometry buffer

• Instrument settings:

  • Excitation: 488 nm laser

  • Emission filter: 530/30 nm bandpass

  • Compensation: Adjust for spectral overlap if using multiple fluorophores

  • Include unstained and isotype controls for gating

• Analysis considerations:

  • Examine both intensity and percentage of positive cells

  • Consider subcellular distribution if using imaging flow cytometry

  • Compare results with Western blot data for validation

This protocol allows for quantitative assessment of INTS3 expression across different cell populations and experimental conditions.

How should researchers interpret changes in INTS3 localization during experimental manipulations?

Changes in INTS3 subcellular localization can provide important insights into its function and regulation:

• Normal INTS3 localization patterns:

  • Primarily nuclear localization with possible nucleolar enrichment

  • Co-localization with sites of active transcription

  • Association with other Integrator complex components

• Interpreting localization changes:

  • Nuclear to cytoplasmic shifts may indicate disrupted nuclear import/export

  • Punctate nuclear distribution may reflect association with specific nuclear bodies

  • Co-localization changes with RNAPII may indicate altered transcriptional regulation

  • Redistribution during cell cycle progression may reflect cell cycle-dependent functions

• Quantification approaches:

  • Nuclear/cytoplasmic ratio measurements

  • Co-localization coefficients with known markers

  • Intensity distribution profiles across defined cellular regions

Research has shown that INTS3 localization and function can be affected by mutations in splicing factors like SRSF2, which has implications for understanding disease mechanisms in leukemia .

How is INTS3 antibody, FITC conjugated being used to investigate INTS3's role in leukemogenesis?

FITC-conjugated INTS3 antibodies have become valuable tools in leukemia research, particularly for investigating the mechanisms linking INTS3 dysfunction to disease progression:

• Protein expression analysis: Studies have demonstrated that INTS3 protein expression is reduced in SRSF2 mutant cells, with further reduction in IDH2/SRSF2 double-mutant contexts . FITC-conjugated INTS3 antibodies enable visualization and quantification of these expression changes.

• Correlation with disease progression: Researchers have used INTS3 antibodies to correlate protein levels with clinical parameters, showing that INTS3 downregulation promotes clonal dominance of Idh2 mutant cells .

• Therapeutic response monitoring: INTS3 antibodies help assess how therapeutic interventions affect INTS3 expression and localization, potentially serving as biomarkers for treatment efficacy.

• Mechanistic studies: Immunofluorescence with INTS3 antibodies helps visualize its interaction with other Integrator subunits, as silencing of INTS3 was associated with reduced protein levels of additional Integrator subunits in SRSF2 mutant AML compared to SRSF2 wild-type AML .

These applications contribute to understanding how aberrant INTS3 splicing and expression contribute to leukemogenesis.

What insights has INTS3 research provided into small nuclear RNA processing in disease contexts?

Research utilizing INTS3 antibodies has revealed important connections between INTS3 dysfunction and snRNA processing abnormalities in disease:

• Altered snRNA cleavage: Studies have shown that SRSF2 single-mutant cells had altered snRNA cleavage similar to those seen with direct INTS3 downregulation, which was exacerbated in IDH2/SRSF2 double-mutant cells .

• Functional consequences: INTS3 encodes a component of the Integrator complex that participates in small nuclear RNA processing in addition to RNAPII pause-release. Dysfunction in these processes contributes to disease pathogenesis .

• Developmental impacts: Attenuation of INTS3 expression in SRSF2 mutant cells caused a blockade of myeloid differentiation, an effect further enhanced in an IDH2 mutant background .

• Disease modeling: Mice transplanted with Idh2 R140Q/+/anti-Ints3 shRNA treated bone marrow cells exhibited myeloid skewing, anemia, and thrombocytopenia, and developed a lethal myelodysplastic syndrome with proliferative features .

FITC-conjugated INTS3 antibodies provide a direct means to visualize these alterations in protein expression that underlie the observed snRNA processing defects.

How can INTS3 antibody research help elucidate the cross-talk between epigenetic regulation and RNA splicing?

INTS3 antibody research has provided crucial insights into the relationship between epigenetic modifications and splicing regulation:

• DNA methylation effects: Studies have revealed that regions of differential DNA hypermethylation significantly overlapped with regions of differential RNA splicing in IDH2 single-mutant and IDH2/SRSF2 double-mutant AML .

• Mechanism identification: Research showed increased DNA methylation at all CpG dinucleotides in the INTS3 exon 4-6 region in IDH2/SRSF2 double-mutant cells compared to control or single-mutant cells .

• Therapeutic implications: Treatment with DNA methyltransferase inhibitors like 5-AZA-CdR significantly reduced RNAPII stalling and decreased aberrant INTS3 splicing, suggesting potential therapeutic approaches .

• Functional validation: Studies using INTS3 minigenes demonstrated that when CG dinucleotides were converted to AT to prevent cytosine methylation, IDH2 mutations no longer promoted mutant SRSF2-mediated intron retention .

These findings identify a pathogenic cross-talk between altered epigenetic state and splicing in a subset of leukemias, with INTS3 at the center of these regulatory mechanisms.

What experimental approaches can researchers use to study INTS3's role in RNA polymerase II regulation?

To investigate INTS3's role in RNAPII regulation, researchers can employ several approaches using FITC-conjugated INTS3 antibodies:

• Chromatin immunoprecipitation (ChIP) assays: INTS3 antibodies can be used to perform ChIP across genomic loci of interest. Research has demonstrated striking accumulation of RNAPII across the INTS3 locus in IDH2/SRSF2 double-mutant cells, revealing important regulatory mechanisms .

• Co-immunoprecipitation studies: Identifying INTS3 protein interactions with RNAPII components and other regulatory factors using pull-down approaches followed by immunoblotting.

• Immunofluorescence co-localization: Using FITC-conjugated INTS3 antibodies alongside antibodies against RNAPII to visualize their spatial relationships during transcription.

• Functional genomics: Combining INTS3 antibody studies with RNA-seq and ChIP-seq to correlate INTS3 binding with RNAPII activity and transcriptional output.

• In vitro transcription assays: Assessing how INTS3 depletion or overexpression affects RNAPII elongation rates and processivity in cell-free systems.

These approaches help elucidate INTS3's role in RNAPII pause-release and its contribution to transcriptional regulation in both normal and disease states.

How does INTS3 dysfunction contribute to myeloid differentiation defects in leukemia?

Research using INTS3 antibodies has revealed important connections between INTS3 dysfunction and myeloid differentiation abnormalities:

• Differentiation blockade: Attenuation of INTS3 expression in SRSF2 mutant cells caused a blockade of myeloid differentiation, an effect further enhanced in an IDH2 mutant background .

• Clonal advantage: Direct Ints3 downregulation in the Idh2 R140Q/+ background resulted in enhanced clonogenic capacity of cells with an immature morphology and immunophenotype .

• Disease phenotype development: Mice transplanted with Idh2 R140Q/+/anti-Ints3 shRNA treated bone marrow cells developed a lethal myelodysplastic syndrome with proliferative features, phenotypes resembling those seen in IDH2/SRSF2 double-mutant mice .

• Molecular mechanisms: INTS3 dysfunction affects snRNA processing and RNAPII regulation, which likely impacts the expression of genes required for proper myeloid differentiation .