UIMC1 Antibody, FITC conjugated

What is UIMC1 and what role does it play in cellular processes?

UIMC1 (Ubiquitin Interaction Motif-Containing Protein 1), also known as RAP80 or BRCA1-A Complex Subunit RAP80, is a ubiquitin-binding protein that specifically recognizes and binds 'Lys-63'-linked ubiquitin chains . It plays a central role in the BRCA1-A complex by specifically binding 'Lys-63'-linked ubiquitinated histones H2A and H2AX at DNA lesion sites, leading to recruitment of the BRCA1-BARD1 heterodimer to sites of DNA damage at double-strand breaks (DSBs) . The BRCA1-A complex also possesses deubiquitinase activity that specifically removes 'Lys-63'-linked ubiquitin on histones H2A and H2AX . Additionally, UIMC1 may indirectly act as a transcriptional repressor by inhibiting the interaction of NR6A1 with the corepressor NCOR1 .

What are the basic characteristics of UIMC1 Antibody, FITC conjugated?

The UIMC1 Antibody, FITC conjugated is a fluorescein isothiocyanate-labeled antibody derived from rabbit that specifically targets human UIMC1 protein . It has the following characteristics:

This FITC-conjugated antibody enables direct visualization of UIMC1 in fluorescence-based applications without requiring secondary antibody labeling .

What are the primary research applications for UIMC1 Antibody, FITC conjugated?

• Flow cytometry for analyzing UIMC1 expression in different cell populations

• Direct immunofluorescence microscopy for visualizing UIMC1 localization

• High-content screening applications

• Multiplexed imaging when combined with antibodies conjugated to spectrally distinct fluorophores

For optimal results, researchers should validate the antibody for their specific application and experimental system, as performance may vary depending on the specific research context .

How should I design validation experiments for UIMC1 Antibody, FITC conjugated?

Validating the UIMC1 Antibody, FITC conjugated before experimental use is critical. A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive controls: Cell lines known to express UIMC1 (testis, ovary, thymus, and heart tissues are recommended based on known tissue specificity)

  • Negative controls: UIMC1 knockout cells or cells with UIMC1 knockdown

• Specificity testing:

  • Blocking experiments with recombinant UIMC1 protein

  • Comparison with other validated UIMC1 antibodies

  • Cross-reactivity assessment with closely related proteins

• Optimal concentration determination:

  • Titration experiments starting at the recommended 1 μg/mL concentration for ELISA applications

  • Adjustment based on signal-to-noise ratio for the specific application

• Validation across multiple techniques when extending beyond ELISA applications, to ensure consistency of results and appropriate performance in each application context.

What are the optimal sample preparation methods for UIMC1 detection?

When preparing samples for UIMC1 detection using the FITC-conjugated antibody, several considerations should be addressed:

• Fixation method:

  • For cellular applications, 4% paraformaldehyde is generally recommended for preserving protein epitopes while maintaining cell morphology

  • Duration and temperature of fixation should be optimized (typically 10-15 minutes at room temperature)

• Permeabilization:

  • Since UIMC1 is a nuclear protein that localizes to sites of DNA damage , sufficient permeabilization is crucial

  • 0.1-0.5% Triton X-100 for 5-10 minutes is a standard starting point

• Blocking:

  • Use 1-5% BSA or 5-10% normal serum from a species different from the antibody host (not rabbit)

  • Include 0.1-0.3% Triton X-100 in blocking solution for nuclear proteins

• Antibody incubation:

  • Start with the recommended dilution range and optimize as needed

  • Consider longer incubation times (overnight at 4°C) for maximum sensitivity

• Special considerations for DNA damage studies:

  • When studying UIMC1 recruitment to DNA damage sites, appropriate DNA damage induction (e.g., irradiation, chemotherapeutic agents) should be performed prior to fixation

  • Timing after damage induction is critical as UIMC1 localization is dynamic

What controls should be included when using UIMC1 Antibody, FITC conjugated?

Proper experimental controls are essential for accurate interpretation of results with UIMC1 Antibody, FITC conjugated:

• Technical controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration as the UIMC1 antibody

  • Secondary antibody-only control (for modified protocols involving secondary enhancement)

  • Autofluorescence control: unstained sample to assess background fluorescence

• Biological controls:

  • Positive tissue/cell control: Samples known to express UIMC1 (testis, ovary, thymus, or heart tissue)

  • Negative tissue/cell control: Samples with minimal or no UIMC1 expression

  • siRNA/shRNA knockdown control: Cells with reduced UIMC1 expression to confirm specificity

  • CRISPR/Cas9 knockout control: Cells with UIMC1 gene deletion (if available)

• Treatment controls for DNA damage response studies:

  • Untreated cells vs. cells with induced DNA damage

  • Time-course samples to track UIMC1 recruitment kinetics

  • ATM/ATR inhibitor treated cells (since UIMC1 is phosphorylated by these kinases upon DNA damage)

How can I optimize the signal-to-noise ratio when using UIMC1 Antibody, FITC conjugated?

Optimizing signal-to-noise ratio is crucial for generating reliable results with FITC-conjugated antibodies:

• Antibody concentration optimization:

  • Perform titration experiments starting with recommended dilution (1 μg/mL for ELISA)

  • Test 2-3 dilutions above and below recommended concentration

  • Select concentration that provides maximum specific signal with minimal background

• Reducing background fluorescence:

  • Use freshly prepared 4% paraformaldehyde for fixation

  • Increase blocking time (2 hours at room temperature or overnight at 4°C)

  • Include 0.1% Tween-20 in wash buffers

  • Filter all solutions to remove particulates that may cause fluorescence artifacts

  • Consider using Sudan Black B (0.1-0.3%) to reduce autofluorescence, particularly in tissue sections

• Photobleaching prevention:

  • Minimize exposure to light during all steps

  • Use antifade mounting media containing DAPI for nuclear counterstaining

  • Capture images promptly after mounting or store slides at 4°C in the dark

• Sample-specific considerations:

  • For tissues with high autofluorescence (brain, liver), consider spectral unmixing during image acquisition

  • For cell lines with low UIMC1 expression, increase antibody incubation time or use signal amplification systems

What are common issues encountered with UIMC1 Antibody, FITC conjugated and how can they be resolved?

Researchers may encounter several challenges when working with FITC-conjugated UIMC1 antibody:

• Weak or absent signal:

  • Possible causes: Insufficient antibody concentration, inadequate permeabilization, epitope masking

  • Solutions: Increase antibody concentration, optimize permeabilization conditions, test different fixation methods, verify UIMC1 expression in sample

• High background:

  • Possible causes: Excessive antibody concentration, insufficient blocking, non-specific binding

  • Solutions: Reduce antibody concentration, increase blocking time/concentration, add 0.1-0.3% Triton X-100 to blocking buffer, increase wash duration/frequency

• Photobleaching:

  • Possible causes: Excessive exposure to light, inadequate mounting medium

  • Solutions: Minimize light exposure during processing, use fresh antifade mounting medium, reduce exposure time during imaging

• Non-specific nuclear staining:

  • Possible causes: Cross-reactivity with other nuclear proteins, excessive antibody concentration

  • Solutions: Increase blocking stringency, reduce antibody concentration, validate with knockout/knockdown controls

• Inconsistent staining across experiments:

  • Possible causes: Variability in fixation/permeabilization, antibody degradation, inconsistent protocol

  • Solutions: Standardize all protocol steps, aliquot antibody to avoid freeze-thaw cycles, prepare fresh reagents for each experiment

How stable is the FITC conjugation and what are the best practices for antibody storage?

The stability of FITC conjugation and appropriate storage are critical for maintaining antibody performance:

• FITC stability considerations:

  • FITC is sensitive to photobleaching and pH changes

  • Optimal pH range for FITC fluorescence is 7.0-9.0

  • Fluorescence intensity may decrease over time, even with proper storage

• Storage recommendations:

  • Store at -20°C for up to 1 year from date of receipt

  • Prepare small aliquots to avoid multiple freeze-thaw cycles

  • Protect from light using amber tubes or by wrapping tubes in aluminum foil

  • Include preservatives as indicated in product formulation (e.g., 0.03% Proclin 300)

• Shipping and temporary storage:

  • Short-term storage (1-2 weeks): 4°C, protected from light

  • Avoid storing diluted antibody solutions for extended periods

  • If working solutions must be stored, add carrier protein (0.1-1% BSA) to prevent adsorption to tube walls

• Monitoring antibody performance:

  • Include positive controls in each experiment to track antibody performance over time

  • Consider refreshing antibody stocks if signal intensity decreases significantly

  • Document lot numbers and performance to identify potential lot-to-lot variability

How can UIMC1 Antibody, FITC conjugated be used to study DNA damage response pathways?

The UIMC1 Antibody, FITC conjugated can be a valuable tool for studying DNA damage response pathways due to UIMC1's critical role in DNA double-strand break repair:

• Visualization of UIMC1 recruitment to DNA damage sites:

  • Combine with markers of DNA double-strand breaks (γH2AX) for co-localization studies

  • Track UIMC1 recruitment kinetics following damage induction using time-lapse imaging

  • Compare UIMC1 recruitment in different cell types or genetic backgrounds

• Analysis of BRCA1 pathway functionality:

  • Assess UIMC1 recruitment in BRCA1-deficient vs. BRCA1-proficient cells

  • Evaluate the impact of cancer-associated BRCA1 mutations on UIMC1 localization

  • Study the interdependence of UIMC1 and other BRCA1-A complex components

• Investigation of ubiquitin signaling in DNA damage:

  • Examine UIMC1 localization in cells treated with proteasome inhibitors

  • Study the relationship between UIMC1 and specific ubiquitin chain types

  • Analyze UIMC1 recruitment following treatment with deubiquitinase inhibitors

• Therapeutic response prediction:

  • Evaluate UIMC1 localization patterns in response to different DNA-damaging therapeutic agents

  • Correlate UIMC1 recruitment dynamics with cellular sensitivity to PARP inhibitors

  • Assess how UIMC1 localization changes in cells developing resistance to DNA-damaging therapies

What strategies can be used to multiplex UIMC1 Antibody, FITC conjugated with other markers in advanced imaging applications?

Multiplexing UIMC1 Antibody, FITC conjugated with other markers can provide rich contextual information about UIMC1 function:

• Spectral compatibility considerations:

  • FITC emission peaks at approximately 519-525 nm (green)

  • Compatible fluorophores for multiplexing include:

    • DAPI/Hoechst (blue) for nuclear counterstaining

    • Cy3/RFP/TRITC (red) for additional markers

    • Cy5/APC (far-red) for additional markers

• Recommended marker combinations:

  • DNA damage pathway analysis:

    • UIMC1-FITC + γH2AX-Cy3 + DAPI

    • UIMC1-FITC + 53BP1-Cy5 + BRCA1-Cy3 + DAPI

  • Ubiquitin signaling analysis:

    • UIMC1-FITC + K63-linked ubiquitin-Cy3 + DAPI

    • UIMC1-FITC + H2A-Cy3 + K63-linked ubiquitin-Cy5 + DAPI

  • Cell cycle analysis:

    • UIMC1-FITC + Cyclin B1-Cy3 + DAPI

    • UIMC1-FITC + EdU-Cy5 (S-phase) + DAPI

• Advanced multiplexing techniques:

  • Sequential staining for higher multiplexing capacity:

    • Image first marker set

    • Strip antibodies using glycine-HCl (pH 2.5) or commercial antibody stripping buffers

    • Re-stain with second marker set

  • Spectral imaging and unmixing to resolve overlapping fluorophore spectra

  • Tyramide signal amplification for enhancing weak signals in multiplexed samples

• Controls for multiplexed imaging:

  • Single-stained controls for each fluorophore to set compensation

  • Fluorescence minus one (FMO) controls to set accurate gates/thresholds

  • Serial dilution of antibodies to ensure no cross-interference

How can UIMC1 Antibody, FITC conjugated be used in conjunction with super-resolution microscopy techniques?

Super-resolution microscopy can reveal detailed spatial organization of UIMC1 at DNA damage sites beyond the diffraction limit:

• Compatibility with super-resolution techniques:

  • Structured Illumination Microscopy (SIM):

    • Compatible with standard FITC fluorophore

    • Achieves ~100-120 nm resolution

    • Allows for live-cell imaging of UIMC1 dynamics

  • Stimulated Emission Depletion (STED):

    • FITC is compatible but not optimal; consider custom conjugation with STED-optimized dyes

    • Can achieve 30-70 nm resolution

    • Best for fixed-cell imaging of UIMC1 organization

  • Single-molecule localization microscopy (STORM/PALM):

    • FITC is not ideal; consider photoconvertible/photoswitchable dyes

    • Can achieve 10-20 nm resolution

    • Powerful for mapping UIMC1 nanoscale organization at damage sites

• Sample preparation adaptations:

  • Thinner samples (≤10 μm) for optimal results

  • Higher quality fixation (fresh 4% PFA, electron microscopy-grade)

  • More stringent background reduction measures

  • Consider mounting in specific media optimized for super-resolution applications

• Advanced research applications:

  • Nanoscale organization analysis:

    • Map precise spatial relationships between UIMC1 and other BRCA1-A complex components

    • Determine clustering patterns of UIMC1 at different time points after damage

    • Analyze changes in UIMC1 organization in different genetic backgrounds

  • Quantitative measurements:

    • Count absolute numbers of UIMC1 molecules recruited to damage sites

    • Measure exact distances between UIMC1 and other damage response factors

    • Track subtle changes in UIMC1 organization during repair progression

How should researchers interpret different patterns of UIMC1 localization?

UIMC1 localization patterns provide important insights into its function and the status of DNA damage response pathways:

• Normal nuclear distribution pattern (undamaged cells):

  • Diffuse nuclear staining with some heterogeneity

  • Exclusion from nucleoli

  • Possible enrichment in certain nuclear domains

• DNA damage-induced foci pattern:

  • Discrete nuclear foci represent UIMC1 recruitment to DNA double-strand breaks

  • Co-localization with γH2AX indicates bona fide DNA damage sites

  • Temporal dynamics: foci typically appear within 5-30 minutes post-damage and resolve within 24-48 hours during successful repair

• Aberrant patterns and their interpretation:

  • Cytoplasmic mislocalization: May indicate defects in nuclear import machinery or UIMC1 mutation

  • Failure to form foci after damage: Suggests dysfunction in upstream ubiquitination events or UIMC1 UIM domains

  • Persistent foci long after damage: May indicate defective DNA repair

  • Diffuse nuclear accumulation without foci: Could reflect non-specific binding or fixation artifacts

• Quantitative interpretation considerations:

  • Number of foci per nucleus correlates with extent of DNA damage

  • Foci size may reflect processing stage of damage sites

  • Intensity of UIMC1 at foci indicates recruitment efficiency

What is the relationship between UIMC1 and other DNA damage response proteins, and how can this be studied?

Understanding UIMC1's relationship with other DNA damage response proteins is crucial for comprehending its role in genomic stability:

• Key UIMC1 interaction partners:

  • BRCA1-A complex components: ABRAXAS1, BRCC3, BABAM1, BRCC45

  • Ubiquitinated histones: H2A-Ub, H2AX-Ub (specifically K63-linked ubiquitin chains)

  • DNA damage signaling proteins: ATM, ATR (which phosphorylate UIMC1)

• Experimental approaches to study interactions:

  • Co-immunoprecipitation: Pull down UIMC1 and detect associated proteins

  • Proximity ligation assay: Detect in situ protein-protein interactions with <40 nm proximity

  • Co-localization analysis: Quantify spatial overlap between UIMC1-FITC and other immunolabeled proteins

  • Sequential ChIP (chromatin immunoprecipitation): Determine co-occupancy on chromatin

• Functional relationship studies:

  • Recruitment dependency experiments: Knockdown one factor and assess UIMC1 localization

  • Domain mutation analysis: Introduce mutations in UIMC1 UIM domains and assess interactions

  • Post-translational modification studies: Evaluate how phosphorylation by ATM/ATR affects UIMC1 interactions

• Data interpretation framework:

  • Temporal sequence: Which factor arrives first at damage sites?

  • Spatial organization: Do factors occupy the same or adjacent space?

  • Dependency relationships: Is recruitment of one factor dependent on another?

  • Functional outcomes: How do interaction disruptions affect repair outcomes?

How can UIMC1 expression and localization data be integrated with clinical and pathological information?

Integrating UIMC1 data with clinical and pathological information can provide valuable insights for translational research:

• Relevance to cancer research:

  • UIMC1 is part of the BRCA1 pathway, which is critical for tumor suppression

  • Alterations in UIMC1 may affect homologous recombination repair efficiency

  • UIMC1 status may influence response to PARP inhibitors and platinum-based chemotherapies

• Integration approaches:

  • Tissue microarray analysis: Correlate UIMC1 expression/localization with tumor type, grade, and stage

  • Patient-derived xenograft models: Compare UIMC1 dynamics in tumors with different treatment responses

  • Multi-omics integration: Correlate UIMC1 protein data with genomic alterations and transcriptomic profiles

• Potential clinical correlations:

  • Treatment response prediction: Does UIMC1 localization pattern predict sensitivity to DNA-damaging agents?

  • Genomic instability assessment: Can UIMC1 foci patterns serve as a biomarker for genomic instability?

  • Synthetic lethality opportunities: Could UIMC1 status identify tumors vulnerable to specific targeted therapies?

• Analytical considerations:

  • Use standardized scoring systems for UIMC1 staining patterns

  • Employ machine learning approaches for unbiased pattern recognition

  • Account for tumor heterogeneity by analyzing multiple regions

  • Consider microenvironment influences on UIMC1 dynamics

What are the limitations of current UIMC1 antibodies and how might these be addressed in future research?

Current UIMC1 antibodies, including FITC-conjugated versions, have several limitations that future research could address:

• Current limitations:

  • Limited application range (primarily ELISA for the FITC-conjugated antibody)

  • Restricted species reactivity (human-specific for the FITC-conjugated version)

  • Potential epitope masking in certain fixation conditions

  • Lack of phospho-specific antibodies to detect activated UIMC1

  • FITC photobleaching limitations in extended imaging experiments

• Future technical improvements:

  • Development of antibodies against different UIMC1 epitopes

  • Creation of phospho-specific antibodies targeting ATM/ATR phosphorylation sites

  • Conjugation with more photostable fluorophores (Alexa Fluor dyes)

  • Generation of monoclonal antibodies for greater specificity

  • Development of nanobodies for super-resolution applications

• Validation approaches for next-generation antibodies:

  • Comprehensive validation using CRISPR/Cas9 knockout controls

  • Cross-validation with orthogonal detection methods

  • Standardized reporting of validation metrics

  • Application-specific performance documentation

What emerging technologies might enhance UIMC1 research beyond antibody-based detection?

Emerging technologies offer exciting opportunities to study UIMC1 biology beyond traditional antibody approaches:

• Genome editing for endogenous tagging:

  • CRISPR/Cas9-mediated knock-in of fluorescent tags (GFP, mCherry) to endogenous UIMC1

  • Split fluorescent protein complementation for detecting protein-protein interactions

  • Degron tagging for rapid protein depletion studies

• Live-cell imaging innovations:

  • Lattice light-sheet microscopy for long-term 3D imaging with minimal phototoxicity

  • 4D imaging of UIMC1 dynamics during DNA damage response

  • Optogenetic tools to control UIMC1 localization or function

• Single-cell analysis technologies:

  • Mass cytometry (CyTOF) for high-parameter analysis of UIMC1 with other markers

  • Single-cell proteomics to analyze UIMC1 levels across heterogeneous populations

  • Spatial transcriptomics to correlate UIMC1 protein data with local gene expression

• Structural biology approaches:

  • Cryo-electron microscopy of UIMC1 in complex with ubiquitinated nucleosomes

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Integrative structural biology combining multiple techniques for complete structural understanding

What are the most promising future research directions for understanding UIMC1 function?

Several promising research directions could significantly advance our understanding of UIMC1 biology:

• Mechanistic studies:

  • Detailed characterization of how UIMC1 recognizes and binds K63-linked ubiquitin chains

  • Investigation of UIMC1's role in regulating transcriptional repression

  • Elucidation of UIMC1 post-translational modifications beyond phosphorylation and sumoylation

  • Understanding the dynamics of BRCA1-A complex assembly and disassembly

• Disease relevance:

  • Comprehensive analysis of UIMC1 alterations across cancer types

  • Evaluation of UIMC1 as a biomarker for DNA repair deficiency

  • Assessment of UIMC1 in aging-related genome instability

  • Investigation of UIMC1 in neurodegenerative diseases involving DNA damage

• Therapeutic opportunities:

  • Exploration of synthetic lethal interactions with UIMC1 deficiency

  • Development of peptide inhibitors targeting UIMC1-ubiquitin interactions

  • Screening for small molecules that modulate UIMC1 function

  • Evaluation of UIMC1 as a predictive biomarker for existing therapies

• Technological developments:

  • High-throughput screening systems to identify UIMC1 modulators

  • AI-powered image analysis tools for quantifying complex UIMC1 localization patterns

  • Patient-derived organoid models to study UIMC1 in a physiologically relevant context

  • Multi-omics integration approaches to place UIMC1 in broader cellular response networks