ITGB3BP Antibody

What is ITGB3BP and what functions should researchers consider when selecting antibodies?

ITGB3BP (also known as Beta3-endonexin, CENP-R, or NRIF3) functions as a transcription coregulator with both coactivator and corepressor capabilities. It plays critical roles in nuclear receptor activity, particularly with retinoid X receptors (RXRs) and thyroid hormone receptors (TRs) . Additionally, ITGB3BP is involved in centromere biology through its interaction with the CENPA-NAC complex, facilitating the incorporation of newly synthesized CENPA into centromeres . The protein also induces apoptosis specifically in breast cancer cells via a caspase-2 mediated pathway . ITGB3BP may additionally function as an inhibitor of cyclin A-associated kinase, further highlighting its multifunctional nature .

When selecting antibodies, researchers must consider which functional domain they wish to study, as different antibodies target different epitopes of the protein. The cellular localization being investigated (chromosome, cytoplasm, nucleus, centromere, or kinetochore) should also dictate antibody selection since ITGB3BP exhibits multiple localizations .

What applications are ITGB3BP antibodies validated for?

ITGB3BP antibodies have been validated for multiple applications based on the search results:

The selection of antibody application should be determined by experimental goals. For protein quantification and molecular weight confirmation, Western blotting is appropriate. For localization studies, immunofluorescence or immunohistochemistry are preferred techniques. When designing experiments, researchers should verify the specific validation status of their chosen antibody for their intended application .

How should researchers choose between monoclonal and polyclonal ITGB3BP antibodies?

The choice between monoclonal and polyclonal antibodies depends on the specific research requirements:

Monoclonal antibodies (such as ABIN565076, clone 3F6) offer:

• Higher specificity for a single epitope

• Reduced background and cross-reactivity

• Greater reproducibility between experiments

• Particularly well-suited for applications requiring high specificity like immunofluorescence

Polyclonal antibodies (such as CAB14859) provide:

• Recognition of multiple epitopes on the target protein

• Enhanced sensitivity for detecting low-abundance proteins

• Better tolerance to protein denaturation and fixation conditions

• Particularly effective for applications like IHC-P

For critical applications, researchers should validate antibody performance in their specific experimental system, regardless of antibody type. When studying specific domains or post-translational modifications, monoclonal antibodies targeting specific epitopes are generally preferable .

What epitope regions are targeted by commonly available ITGB3BP antibodies?

Various commercial antibodies target different epitope regions of ITGB3BP:

The choice of epitope region can significantly impact experimental outcomes. Antibodies targeting functional domains may interfere with protein-protein interactions. N-terminal or C-terminal targeting antibodies might not detect truncated isoforms or proteins with post-translational modifications at those regions .

What are the optimal storage and handling conditions for ITGB3BP antibodies?

To maintain optimal antibody performance, researchers should follow these guidelines:

• Storage temperature: Most ITGB3BP antibodies should be stored at -20°C for long-term storage, with aliquoting recommended to avoid freeze-thaw cycles

• Working dilutions: For the rabbit polyclonal antibody CAB14859, a dilution range of 1:50 to 1:200 is recommended for IHC-P applications

• Avoiding contamination: Use sterile techniques when handling antibodies to prevent microbial contamination

• Centrifugation: Brief centrifugation before opening vials is recommended to collect solution at the bottom of the tube

• Buffer compatibility: Verify compatibility with intended buffers; some antibodies may have specific buffer requirements

Researchers should always consult manufacturer-specific recommendations as conditions may vary between suppliers and antibody formulations .

What methods can be used to validate the specificity of ITGB3BP antibodies?

Rigorous validation of ITGB3BP antibodies is essential for reliable research findings. Several complementary approaches are recommended:

Genetic validation methods:

• siRNA or shRNA knockdown of ITGB3BP, followed by Western blotting or immunostaining to confirm signal reduction

• CRISPR/Cas9-mediated knockout of ITGB3BP as the gold standard for specificity confirmation

• Overexpression systems using tagged ITGB3BP constructs to verify antibody detection

Biochemical validation methods:

• Pre-adsorption tests with the immunizing peptide or recombinant protein (particularly the GST-tagged recombinant ITGB3BP used as immunogen in ABIN565076)

• Testing antibody recognition on recombinant ITGB3BP protein with known concentration

• Western blot analysis to confirm detection of a band at the expected molecular weight (approximately 20kDa)

Comparative validation methods:

• Parallel testing with multiple independent antibodies targeting different ITGB3BP epitopes

• Comparison with GFP-tagged ITGB3BP localization patterns

• Cross-validation using orthogonal detection methods (e.g., mass spectrometry)

The Human Protein Atlas utilizes enhanced validation methods including siRNA knockdown, GFP-tagged cell lines, and independent antibodies targeting different epitopes to confirm antibody specificity .

What controls should be included when using ITGB3BP antibodies in experimental protocols?

Robust experimental design requires appropriate controls:

Positive controls:

• Cell lines with confirmed ITGB3BP expression (based on RNA-seq or proteomics data)

• Recombinant ITGB3BP protein (such as the partial recombinant protein with GST tag used as immunogen for ABIN565076)

• Tissues with known expression patterns (according to Human Protein Atlas data)

Negative controls:

• ITGB3BP-knockout or knockdown samples

• Isotype control antibodies matched to the primary antibody (e.g., IgG2a for ABIN565076)

• Primary antibody omission controls

• Non-expressing tissues or cell lines

Technical controls:

• Loading controls for Western blotting (e.g., housekeeping proteins)

• Internal staining controls for immunohistochemistry and immunofluorescence

• Dilution series to determine optimal antibody concentration

• Secondary antibody-only controls to assess non-specific binding

Researchers should document all control results thoroughly to support the validity of experimental findings with ITGB3BP antibodies .

How can researchers optimize ITGB3BP antibody conditions for subcellular localization studies?

ITGB3BP localizes to multiple cellular compartments including chromosomes, cytoplasm, nucleus, centromeres, and kinetochores . Optimizing detection in each location requires specific considerations:

Fixation methods:

• For nuclear/chromosomal localization: 4% paraformaldehyde fixation preserves nuclear structure

• For cytoplasmic localization: Methanol fixation may provide better cytoplasmic protein retention

• For centromere/kinetochore detection: Combined paraformaldehyde/methanol protocols may be optimal

Permeabilization optimization:

• Nuclear access often requires stronger permeabilization (0.5% Triton X-100)

• Cytoplasmic epitopes may require gentler permeabilization (0.1-0.2% Triton X-100)

• Testing multiple detergents (Triton X-100, NP-40, saponin) at varying concentrations

Antibody dilution and incubation:

• Starting with manufacturer recommendations (e.g., 1:50-1:200 for IHC-P with CAB14859)

• Testing longer incubation times (overnight at 4°C) for weak signals

• Evaluating blocking reagents to improve signal-to-noise ratio

Co-localization studies:

• Combining ITGB3BP antibody with established centromere/kinetochore markers

• Using super-resolution microscopy for precise localization

• Sequential staining protocols when antibody species conflicts occur

Systematic optimization should document all variables (fixation, permeabilization, blocking, antibody dilution, incubation time/temperature) to establish reproducible protocols .

What are effective troubleshooting approaches for weak or non-specific ITGB3BP antibody staining?

Researchers frequently encounter staining issues with ITGB3BP antibodies that can be addressed through systematic troubleshooting:

For weak or absent signals:

• Verify protein expression in the sample through alternative methods (RT-PCR, RNA-seq)

• Increase antibody concentration (starting with manufacturer recommendations like 1:50-1:200)

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

• Test alternative epitope retrieval methods for fixed samples

• Evaluate different detection systems (e.g., amplification with tyramide signal amplification)

• Confirm antibody functionality with positive controls

For high background or non-specific staining:

• Increase blocking duration and concentration (BSA, normal serum matched to secondary antibody species)

• Optimize washing steps (increase number, duration, or detergent concentration)

• Reduce primary and secondary antibody concentrations

• Pre-adsorb secondary antibodies with tissue powder from the species being studied

• Test alternative fixation protocols that may better preserve epitope structure

• Consider switching to a more specific monoclonal antibody like ABIN565076

For inconsistent results:

• Prepare larger antibody aliquots to minimize freeze-thaw cycles

• Standardize all protocol components including buffers and reagent lots

• Implement positive and negative controls in every experiment

• Document exact incubation times and temperatures

• Consider batch processing of samples for comparative studies

Researchers should systematically modify one variable at a time while maintaining detailed records of optimization attempts .

How can ITGB3BP antibodies be effectively used in cancer research applications?

ITGB3BP plays significant roles in cancer biology, particularly through its ability to induce apoptosis in breast cancer cells via a caspase-2 mediated pathway . Researchers can optimize ITGB3BP antibody use in cancer studies through several approaches:

Expression analysis in cancer tissues:

• Use immunohistochemistry with antibodies like CAB14859 (dilution 1:50-1:200) on cancer tissue microarrays

• Compare ITGB3BP expression between normal and malignant tissues

• Correlate expression patterns with clinical parameters and survival data

• Analyze subcellular localization changes in cancer progression

Functional studies:

• Combine ITGB3BP antibodies with apoptosis markers to study its pro-apoptotic function

• Investigate the relationship between ITGB3BP and caspase-2 activation using co-immunoprecipitation

• Examine ITGB3BP's interaction with integrin signaling in cancer cell migration and invasion

• Study ITGB3BP's role in centromere function and chromosomal stability in cancer cells

Therapeutic target validation:

• Use antibodies to assess ITGB3BP expression before and after treatment

• Correlate ITGB3BP levels with therapy response

• Investigate ITGB3BP's interaction with nuclear receptors (RXRs, TRs) in hormone-responsive cancers

Technical considerations:

• Include cancer-specific positive and negative controls

• Validate antibody specificity in each cancer cell line studied

• Consider isoform-specific detection using epitope-specific antibodies

• Implement multiplexed immunofluorescence to study ITGB3BP in the context of tumor heterogeneity

Researchers should select antibodies based on the specific aspect of ITGB3BP biology being investigated in cancer research .

What are the considerations for detecting specific ITGB3BP isoforms and variants?

ITGB3BP has multiple isoforms with distinct functions. For instance, isoform 1 is involved in the coactivation of nuclear receptors for retinoid X (RXRs) and thyroid hormone (TRs), while other isoforms lack this function . Researchers must consider several factors when designing isoform-specific detection strategies:

Antibody epitope selection:

• Choose antibodies targeting regions specific to the isoform of interest

• For isoform 1-specific detection, select antibodies against domains involved in nuclear receptor interactions

• Avoid antibodies targeting common regions when isoform specificity is required

Validation approaches for isoform specificity:

• Express recombinant isoforms individually and test antibody recognition

• Use isoform-specific siRNAs to confirm antibody specificity

• Perform Western blotting to verify detection of the correct molecular weight variant

Experimental design considerations:

• Use RT-PCR with isoform-specific primers as a complementary approach

• Consider mass spectrometry for unambiguous isoform identification

• Design co-immunoprecipitation experiments to identify isoform-specific interaction partners

Technical challenges:

• Small size differences between isoforms may be difficult to resolve by standard Western blotting

• Post-translational modifications may affect antibody recognition

• Differential localization of isoforms may require compartment-specific extraction methods

The use of multiple antibodies targeting different epitopes can provide complementary data when studying ITGB3BP isoforms. For example, combining antibodies like ABIN565076 (AA 1-100) with others targeting different regions can help confirm isoform-specific findings .

What are the optimal protocols for ITGB3BP co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is valuable for studying ITGB3BP interactions with proteins like CENPA-NAC complex components or nuclear receptors. The following protocol outlines key considerations:

Lysis buffer optimization:

• Use non-denaturing buffers to preserve protein-protein interactions

• For nuclear interactions: 20mM HEPES pH 7.9, 150mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 0.5% NP-40, 10% glycerol

• For centromere/kinetochore interactions: Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Add protease inhibitor cocktails to prevent protein degradation

Antibody selection:

• Choose antibodies validated for immunoprecipitation applications

• Consider using monoclonal antibodies like ABIN565076 for higher specificity

• Use 2-5μg of antibody per mg of protein lysate

Pre-clearing and controls:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Include isotype-matched IgG controls (e.g., mouse IgG2a for ABIN565076)

• Consider using ITGB3BP-depleted lysates as negative controls

Elution and detection:

• Gentle elution with peptide competition for downstream functional assays

• Standard SDS elution for Western blot analysis

• Analyze eluates by Western blotting or mass spectrometry

Verification approaches:

• Perform reverse Co-IP experiments

• Validate interactions with alternative methods (proximity ligation assay, FRET)

• Use truncation mutants to map interaction domains

Researchers should optimize Co-IP conditions for each specific interaction partner being studied with ITGB3BP .

How can researchers effectively use ITGB3BP antibodies in chromatin immunoprecipitation (ChIP) experiments?

Given ITGB3BP's role as a transcriptional coregulator , ChIP experiments are valuable for understanding its genomic targets:

Sample preparation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• For studying centromere association, optimize crosslinking conditions (consider dual crosslinkers like DSG followed by formaldehyde)

• Sonicate chromatin to 200-500bp fragments (verify fragment size by gel electrophoresis)

Antibody selection and immunoprecipitation:

• Select antibodies with minimal background binding to DNA

• Use 2-5μg of antibody per ChIP reaction

• Include appropriate controls (input, IgG, positive control locus)

Washing and elution:

• Implement stringent washing steps to minimize background

• Elute DNA-protein complexes and reverse crosslinks

• Purify DNA for downstream analysis

Data analysis approaches:

• qPCR for candidate target regions

• ChIP-seq for genome-wide binding profile

• Integrate with RNA-seq to correlate binding with gene expression

• Consider sequential ChIP (ChIP-reChIP) to study co-occupancy with known interaction partners

Validation strategies:

• Compare binding at regions with predicted binding motifs

• Correlate binding with expression changes after ITGB3BP knockdown

• Confirm binding site specificity through reporter assays

Researchers should pay particular attention to known ITGB3BP functions in transcriptional regulation when designing ChIP experiments, especially its interactions with nuclear receptors like RXRs and TRs .

What quantitative techniques are recommended for analyzing ITGB3BP expression levels?

Multiple approaches can be used for quantitative analysis of ITGB3BP expression:

Western blot quantification:

• Use recombinant ITGB3BP protein standards for absolute quantification

• Include appropriate loading controls (β-actin, GAPDH, tubulin)

• Analyze band intensity with software like ImageJ, normalizing to loading controls

• Consider the calculated molecular weight of approximately 20kDa

Immunohistochemistry quantification:

• Score staining intensity categories (0, 1+, 2+, 3+)

• Determine percentage of positive cells

• Calculate H-scores or other semi-quantitative metrics

• Use automated image analysis for more objective quantification

Flow cytometry:

• Optimize fixation and permeabilization for intracellular staining

• Include fluorescence-minus-one (FMO) controls

• Report data as mean fluorescence intensity (MFI) or percent positive

• Consider dual staining with cell cycle markers to analyze cell cycle-dependent expression

qPCR and digital PCR:

• Design primers specific to ITGB3BP transcripts

• Use absolute quantification with standard curves

• Normalize to validated reference genes

• Consider isoform-specific quantification

Mass spectrometry:

• Use targeted approaches (SRM/MRM) for absolute quantification

• Analyze ITGB3BP-specific peptides for relative quantification

• Include isotope-labeled standards for accurate quantification

Researchers should select quantification methods based on their specific research questions and available resources .

How can researchers utilize ITGB3BP antibodies in single-cell analysis techniques?

With the growing importance of single-cell technologies, ITGB3BP antibodies can be adapted for these applications:

Single-cell immunofluorescence:

• Optimize antibody dilutions for single-cell sensitivity

• Implement signal amplification methods for low-abundance detection

• Combine with other markers for multiparameter analysis

• Use quantitative image analysis to measure cell-to-cell variation

Mass cytometry (CyTOF):

• Conjugate ITGB3BP antibodies with rare earth metals

• Include in multiplexed panels with other centromere/kinetochore markers

• Analyze data with dimensionality reduction techniques (tSNE, UMAP)

• Correlate ITGB3BP levels with cell cycle or differentiation markers

Single-cell Western blotting:

• Adapt ITGB3BP antibody protocols for microfluidic single-cell Western platforms

• Optimize antibody concentrations for reduced volumes

• Correlate protein levels with phenotypic markers at single-cell resolution

Spatial transcriptomics integration:

• Combine ITGB3BP immunostaining with spatial transcriptomics methods

• Correlate protein localization with local transcriptional programs

• Analyze tissue microenvironments for ITGB3BP expression patterns

Researchers should carefully validate antibodies for single-cell applications, as sensitivity and specificity become even more critical at this resolution .

What considerations apply when using ITGB3BP antibodies in high-throughput screening applications?

High-throughput screening with ITGB3BP antibodies requires special optimization:

Assay miniaturization:

• Adapt standard protocols for microplate formats (96, 384, or 1536-well)

• Determine minimum antibody concentrations that maintain signal-to-noise ratios

• Optimize fixation, permeabilization, and washing steps for automation

Automation compatibility:

• Select antibodies with robust performance across batches

• Develop protocols compatible with liquid handling systems

• Implement quality control measures for antibody performance across plates

Multiplexed detection strategies:

• Combine ITGB3BP antibodies with other markers of interest

• Select fluorophores with minimal spectral overlap

• Consider sequential staining approaches for antibodies from the same species

Data analysis pipelines:

• Implement automated image analysis for phenotypic screening

• Develop robust segmentation algorithms for nuclear/cytoplasmic localization

• Create analysis pipelines that quantify ITGB3BP levels, localization, and correlation with phenotypes

Validation in screening context:

• Include positive and negative controls on each plate

• Calculate Z' factors to assess assay quality

• Validate hits with orthogonal approaches

Researchers conducting high-throughput screens should perform extensive validation of ITGB3BP antibodies under their specific screening conditions .

What are the best practices for using ITGB3BP antibodies in studying centromere and kinetochore biology?

ITGB3BP's role in centromere biology through CENPA-NAC complex interactions makes it valuable for studying these structures:

Sample preparation considerations:

• Optimize cell synchronization to enrich for mitotic cells

• Use specific fixation methods that preserve centromere/kinetochore structures

• Consider chromosome spreading techniques for improved resolution

Co-localization studies:

• Combine ITGB3BP antibodies with established centromere markers (CENP-A, CENP-B)

• Use kinetochore markers (HEC1, CENP-E) to distinguish centromere vs. kinetochore localization

• Implement super-resolution microscopy (STORM, STED, SIM) for detailed localization

Functional analysis approaches:

• Correlate ITGB3BP localization with cell cycle stages

• Analyze recruitment dynamics during centromere assembly

• Study effects of ITGB3BP depletion on centromere/kinetochore integrity

Technical optimization:

• Test both monoclonal and polyclonal antibodies for optimal detection

• Consider antigen retrieval methods specific to centromeric chromatin

• Implement quantitative image analysis for measuring ITGB3BP enrichment at centromeres

Experimental controls:

• Include mitotic index measurements to normalize for cell cycle effects

• Use ITGB3BP knockdown cells as specificity controls

• Compare with other CENP proteins for validation of centromeric localization

Researchers should leverage ITGB3BP's known cellular localization patterns to optimize detection protocols specifically for centromere/kinetochore biology .

How are advances in antibody technology improving ITGB3BP research?

Emerging antibody technologies are enhancing ITGB3BP research capabilities:

Recombinant antibody development:

• Increased reproducibility through defined sequence and production

• Reduced batch-to-batch variation compared to traditional methods

• Enhanced antibody engineering possibilities for specialized applications

• Potential for renewable sources without animal immunization

Advanced detection systems:

• Multiplexed detection using spectral unmixing

• Signal amplification technologies for low-abundance detection

• Proximity-based detection methods for studying protein-protein interactions

• Integration with mass spectrometry for proteomic profiling

Spatial biology applications:

• Highly multiplexed imaging approaches (e.g., CODEX, Hyperion)

• In situ sequencing combined with protein detection

• 3D imaging with tissue clearing techniques

• Spatial transcriptomics correlation with protein localization

Computationally aided analysis:

• Machine learning approaches for pattern recognition in localization studies

• Automated quantification of expression levels across tissues

• Systems biology integration of protein expression with other -omics data

Researchers should stay informed about these technological advances to enhance their ITGB3BP studies while maintaining rigorous validation practices .

What are the emerging applications of ITGB3BP antibodies in interdisciplinary research?

ITGB3BP antibodies are finding applications across diverse research fields:

Cancer biology:

• Biomarker development based on ITGB3BP's pro-apoptotic function in breast cancer

• Therapeutic target validation in combination with genomic and transcriptomic data

• Correlation with chromosomal instability phenotypes in cancer progression

• Patient stratification based on ITGB3BP expression patterns

Developmental biology:

• Tracking centromere assembly during early development

• Studying ITGB3BP's role in nuclear receptor signaling during differentiation

• Investigating chromosomal stability mechanisms during rapid cell divisions

Regenerative medicine:

• Monitoring chromosomal integrity in stem cell populations

• Assessing nuclear receptor activity in differentiating cells

• Evaluating centromere function in induced pluripotent stem cells

Precision medicine applications:

• Development of companion diagnostics for therapies targeting pathways involving ITGB3BP

• Integration with multi-omics approaches for comprehensive patient profiling

• Correlation of ITGB3BP status with treatment response