CSRNP3 Antibody, FITC conjugated

What is CSRNP3 and what cellular functions does it serve?

CSRNP3 (Cysteine-Serine-Rich Nuclear Protein 3) is a member of the CSRNP family of nuclear proteins with potential transcription factor activity. The protein contains a conserved cysteine-rich domain and is predominantly localized in the nucleus, as confirmed by immunofluorescence staining that overlaps with DAPI nuclear staining . CSRNP3 migrates at an apparent molecular weight of approximately 95 kDa in SDS-PAGE, which is larger than its predicted size of 66 kDa, likely due to the acidic nature of the protein . While CSRNP3 alone does not show transactivation activity in 293T cells, it does demonstrate transactivation capabilities in yeast reporter strains, suggesting context-dependent transcriptional activity .

CSRNP3 appears to play roles in immune response pathways, with functional enrichment analysis positively associating CSRNP proteins with acute inflammatory response and humoral immune response pathways . The protein has also been implicated as part of a prognostic biomarker signature in certain disease contexts .

What are the key specifications of commercially available CSRNP3 Antibody, FITC conjugated?

Commercial CSRNP3 Antibody, FITC conjugated products typically have the following specifications:

These antibodies are designed for research use only (RUO) and are available in various sizes (typically 50-200 µl) .

How does CSRNP3 relate to other members of the CSRNP family?

CSRNP3 is one of three members of the cysteine-serine-rich nuclear protein family, alongside CSRNP1 and CSRNP2. These three proteins share conserved structural features:

• All three proteins have similar gene structures with corresponding exon-intron boundaries .

• They share a conserved cysteine-rich domain in the amino-terminal portion .

• All three family members exhibit nuclear localization .

• Molecular weight: CSRNP1 appears as multiple bands with predominant forms at 90 and 100 kDa, CSRNP2 as a single band at 80 kDa, and CSRNP3 as a single band at 95 kDa .

• Transcriptional activity: CSRNP1 shows strong transactivation activity (70-fold higher than control) in 293T cells, while CSRNP2 and CSRNP3 do not demonstrate this activity in 293T cells but do show activity in yeast reporter strains .

• Expression patterns and prognostic implications: In certain disease contexts, CSRNP1 and CSRNP3 expression is lower in high-risk groups while CSRNP2 expression is higher .

The three proteins may have both overlapping and distinct functions in immune regulation, with differential associations with various immune cell types in normal versus diseased tissues .

What are the validated applications for CSRNP3 Antibody, FITC conjugated?

CSRNP3 Antibody, FITC conjugated has been validated for several research applications:

• Immunofluorescence (IF): The FITC conjugation makes this antibody particularly suitable for direct immunofluorescence studies without requiring secondary antibody detection . This application is useful for studying protein localization, which is particularly relevant given CSRNP3's nuclear localization.

• Flow Cytometry: The FITC conjugation enables direct detection in flow cytometry applications, facilitating quantitative analysis of CSRNP3 expression in cell populations.

• Immunocytochemistry (ICC): CSRNP3 Antibody can be used to detect the protein in cultured cells, which helps determine subcellular localization and expression patterns .

• Immunohistochemistry (IHC): Though not the primary application for FITC-conjugated antibodies (which are more suited to fluorescence microscopy), these antibodies may be used in frozen tissue sections for IHC applications .

For optimal results, researchers should determine the appropriate dilutions empirically for their specific experimental systems .

How should I design experiments to study CSRNP3's role in immune responses?

Based on the known associations between CSRNP3 and immune responses, the following experimental design approach is recommended:

• Cell Type Selection: Focus on immune cell types that show significant correlation with CSRNP3 expression, particularly:

  • Type 2 T helper cells

  • Mast cells

  • Natural killer cells
    These cell types have shown positive association with CSRNP expression in certain tissues .

• Comparative Analysis: Design experiments that compare normal versus diseased tissue/cells, as the immune infiltration profiles of CSRNP proteins differ between these conditions .

• Methodology Pipeline:

  • Begin with expression analysis using RT-qPCR and western blotting to confirm CSRNP3 levels

  • Use FITC-conjugated CSRNP3 antibody for immunofluorescence to visualize protein localization

  • Perform co-localization studies with markers for specific immune cell types

  • Conduct functional studies through knockdown/knockout approaches followed by immune response assays

  • Consider immune cell isolation and co-culture experiments to assess direct effects

• Controls:

  • Include both positive controls (tissues/cells known to express CSRNP3)

  • Use isotype controls for antibody specificity

  • Consider examining all three CSRNP family members simultaneously for comparative analysis

• Readouts: Measure cytokine production, immune cell infiltration, and signaling pathway activation as functional readouts of CSRNP3's impact on immune responses.

What are the optimal sample preparation methods for CSRNP3 immunofluorescence staining?

For optimal immunofluorescence staining with CSRNP3 Antibody, FITC conjugated:

• Cell Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash 3 times with PBS

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes (critical for nuclear proteins like CSRNP3)

  • Alternatively, use cold methanol (-20°C) for 10 minutes for simultaneous fixation and permeabilization

• Blocking:

  • Block with 5% normal serum (from the same species as the secondary antibody would be if not using directly conjugated antibodies) in PBS with 0.1% Tween-20 for 1 hour at room temperature

  • For directly conjugated antibodies like CSRNP3-FITC, use serum from the same species as the host (rabbit in this case)

• Antibody Incubation:

  • Dilute CSRNP3 Antibody, FITC conjugated in blocking buffer (optimal dilution should be determined empirically, typically starting at 1:50-1:200)

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3 times with PBS-T (PBS with 0.1% Tween-20)

• Nuclear Counterstaining:

  • Since CSRNP3 is a nuclear protein, counterstain with DAPI (1 μg/mL) for 5 minutes

  • This allows co-localization assessment as CSRNP3 has been shown to overlap with DAPI staining

• Mounting and Imaging:

  • Mount slides with anti-fade mounting medium

  • When imaging, be aware that FITC may photobleach quickly; minimize exposure and consider using anti-fade reagents

  • Capture images using appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm)

• Controls:

  • Include a negative control (no primary antibody)

  • Consider using cells with verified low or no CSRNP3 expression as additional negative controls

What are common issues with CSRNP3-FITC antibody staining and how can they be resolved?

How can I validate the specificity of CSRNP3 Antibody, FITC conjugated in my experimental system?

To ensure the specificity of CSRNP3 Antibody, FITC conjugated, implement the following validation steps:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess blocking peptide (the immunogen used to generate the antibody)

  • In parallel, use untreated antibody on identical samples

  • A significant reduction in signal with the peptide-blocked antibody confirms specificity

• Genetic Validation:

  • Test the antibody in CSRNP3 knockout/knockdown models

  • Use siRNA or CRISPR-Cas9 to reduce CSRNP3 expression

  • Compare staining patterns between wild-type and CSRNP3-depleted samples

  • Previous studies have utilized gene-deficient mice for each CSRNP family member

• Cross-Reactivity Assessment:

  • Test the antibody on samples from species outside the claimed reactivity

  • Verify reactivity across the stated species (human, mouse, rat) if relevant to your research

• Multiple Antibody Validation:

  • Compare results with non-conjugated CSRNP3 antibodies or those from different manufacturers

  • Use antibodies targeting different epitopes of CSRNP3

  • Consistent patterns across different antibodies strengthen confidence in specificity

• Western Blot Validation:

  • Perform Western blot to confirm the antibody recognizes a protein of the expected molecular weight (~95 kDa)

  • Look for a single predominant band at the appropriate size

• Correlation with mRNA Expression:

  • Compare antibody staining intensity with CSRNP3 mRNA levels measured by qPCR

  • Positive correlation supports antibody specificity

• Mass Spectrometry Validation:

  • For advanced validation, perform immunoprecipitation followed by mass spectrometry

  • Confirm the pulled-down protein is indeed CSRNP3

What controls should I include when using CSRNP3 Antibody, FITC conjugated for flow cytometry?

For rigorous flow cytometry experiments using CSRNP3 Antibody, FITC conjugated, the following controls are essential:

• Unstained Control:

  • Cells processed identically but without any antibody

  • Establishes baseline autofluorescence and sets negative population gates

• Isotype Control:

  • FITC-conjugated rabbit IgG (matching the CSRNP3 antibody's host and isotype) at the same concentration

  • Controls for non-specific binding due to Fc receptors or other non-specific interactions

  • Should be from the same manufacturer when possible for comparable conjugation methods

• Fluorescence Minus One (FMO) Controls:

  • For multicolor panels, samples with all fluorochromes except FITC

  • Helps identify spillover effects and set proper gates

• Positive Control Samples:

  • Cells known to express CSRNP3 at high levels

  • Useful for setting positive gates and confirming antibody functionality

• Negative Control Samples:

  • Cells known to express little or no CSRNP3

  • CSRNP3 knockdown or knockout cells if available

• Viability Dye:

  • Include a viability dye compatible with FITC (non-overlapping emission spectrum)

  • Excludes dead cells which can bind antibodies non-specifically

• Fixation Controls:

  • If cells are fixed and permeabilized (necessary for nuclear proteins like CSRNP3):

    • Include controls processed identically but without permeabilization

    • This helps assess the contribution of membrane-bound versus intracellular staining

• Dilution Series:

  • During optimization, test a range of antibody concentrations

  • Helps identify the optimal signal-to-noise ratio

  • Typically start with manufacturer's recommendation and test 2-fold dilutions up and down

• Compensation Controls:

  • Single-color controls for each fluorochrome in your panel

  • Essential for accurate compensation in multicolor experiments

How can CSRNP3 expression patterns be correlated with immune cell infiltration in tissues?

To investigate correlations between CSRNP3 expression and immune cell infiltration:

• Multiplex Immunofluorescence Approach:

  • Perform multiplex staining with CSRNP3-FITC alongside markers for specific immune cell populations

  • Based on previous findings, focus particularly on:

    • Type 2 T helper cells (using markers like GATA3, ST2)

    • Mast cells (using tryptase, c-Kit)

    • Natural killer cells (using CD56, NKp46)

    • CD56 bright natural killer cells (using CD56bright, CD16dim)

    • Activated CD8 T cells (using CD8, CD69)

  • These cell types have shown significant correlations with CSRNP expression

• Digital Pathology Analysis:

  • Use automated scanning and analysis software to quantify:

    • CSRNP3 expression levels (intensity of FITC signal)

    • Density of various immune cell populations

    • Co-localization patterns

  • Perform spatial analysis to determine proximity relationships

• Correlation Analysis Methodology:

  • Calculate Pearson or Spearman correlation coefficients between CSRNP3 expression and immune cell counts

  • Perform multivariate analysis to account for confounding factors

  • Consider tissue-specific differences, as CSRNP3 correlations with immune cells differ between normal and diseased tissues

• Single-Cell Approaches:

  • Complement tissue analysis with single-cell RNA sequencing to precisely identify cell types expressing CSRNP3

  • Perform CyTOF (mass cytometry) analysis with metal-tagged antibodies against CSRNP3 and immune cell markers

  • These approaches allow higher-dimensional analysis of correlations

• Intervention Studies:

  • Manipulate CSRNP3 expression using overexpression or knockdown approaches

  • Assess changes in immune cell recruitment and activation

  • This helps establish causality beyond correlation

Research has shown that CSRNP3 is positively associated with type 2 T helper cells, mast cells, and natural killer cells, while negatively associated with CD56 bright natural killer cells and activated CD8 T cells in certain tissues . These patterns differ between normal and diseased states, suggesting context-dependent roles.

What are the methodological considerations for investigating the role of CSRNP3 in transcriptional regulation?

Given that CSRNP3 is a potential transcription factor with nuclear localization , the following methodological approaches are recommended:

• Chromatin Immunoprecipitation (ChIP) Studies:

  • Use CSRNP3 antibodies to perform ChIP followed by sequencing (ChIP-seq)

  • Consider using unfixed ChIP protocols as the FITC conjugate may interfere with standard formaldehyde fixation

  • Alternative approach: Express tagged versions of CSRNP3 (FLAG, HA) for ChIP using tag antibodies

  • Analyze binding motifs and genomic regions to identify direct target genes

  • Compare with known transcription factor binding sites

• Transcriptional Reporter Assays:

  • Design luciferase reporter constructs with promoters of potential target genes

  • Previous studies have used GAL4 fusion proteins to test CSRNP transactivation

  • Test the effect of CSRNP3 overexpression or knockdown on reporter activity

  • Include controls with mutated binding sites to confirm specificity

• Protein-Protein Interaction Studies:

  • Identify CSRNP3 co-factors using co-immunoprecipitation followed by mass spectrometry

  • Perform proximity ligation assays to visualize interactions in situ

  • Consider BioID or APEX2 proximity labeling to identify the CSRNP3 protein interaction network

• DNA-Binding Studies:

  • Perform electrophoretic mobility shift assays (EMSA) to test direct DNA binding

  • Use recombinant CSRNP3 protein with candidate target sequences

  • Include competition assays and supershift with CSRNP3 antibodies

• Gene Expression Profiling:

  • Compare transcriptomes after CSRNP3 modulation (overexpression/knockdown)

  • Perform RNA-seq analysis to identify global changes in gene expression

  • Focus analysis on genes involved in immune pathways, based on CSRNP3's known associations

• Domain Analysis:

  • Create deletion mutants to identify functional domains

  • Previous research mapped the transactivation domain of the related CSRNP1 to the last ninety amino acids

  • Test these constructs in reporter assays to determine critical regions for CSRNP3 function

• Context-Dependent Activity:

  • Test CSRNP3 activity in multiple cell types, as findings show CSRNP3 has transactivation activity in yeast but not in 293T cells

  • Investigate cofactors that might explain this context-dependency

How might CSRNP3 antibodies be used in investigating disease biomarkers and prognostic indicators?

Building on findings that the CSRNP gene family serves as prognostic biomarkers in certain conditions , advanced research applications include:

• Tissue Microarray (TMA) Analysis:

  • Develop standardized immunohistochemistry or immunofluorescence protocols using CSRNP3-FITC antibodies

  • Apply to TMAs containing multiple patient samples with clinical follow-up data

  • Quantify CSRNP3 expression using digital pathology and correlate with:

    • Clinical outcomes (survival, recurrence)

    • Treatment response

    • Disease stage and progression

• Multivariate Prognostic Model Development:

  • Combine CSRNP3 expression with other biomarkers

  • Previous research established a prognostic risk score: Risk score = 0.754 × exp CSRNP1 + 0.820 × exp CSRNP3 - 1.428 × exp CSRNP2

  • Develop and validate similar models for specific diseases

  • Assess the added value of CSRNP3 over existing clinical markers

• Liquid Biopsy Applications:

  • Investigate CSRNP3 protein or mRNA in circulating tumor cells or exosomes

  • Develop assays to detect CSRNP3 in blood or other accessible fluids

  • Assess correlation with tissue expression and clinical outcomes

• Integration with Genomic and Epigenomic Data:

  • Correlate CSRNP3 expression with:

    • Mutation burden (CSRNP1 and CSRNP3 expression associated with better prognosis in both high and low mutant burden groups)

    • DNA methylation status (specific methylation sites like cg07811002 affect CSRNP3 expression and outcomes)

    • Copy number alterations

  • Perform integrated multi-omics analysis

• Therapeutic Response Prediction:

  • Assess CSRNP3 expression as a predictor of response to:

    • Immunotherapies (given CSRNP3's association with immune cell types)

    • Targeted therapies

    • Conventional chemotherapies

  • Develop companion diagnostic approaches using standardized CSRNP3 immunostaining

• Mechanistic Studies:

  • Investigate how CSRNP3 mechanistically contributes to disease progression

  • Focus on its immune regulatory functions and transcriptional activity

  • Determine if CSRNP3 could serve as a therapeutic target itself

• Methodological Standardization:

  • Develop reference standards for CSRNP3 quantification

  • Establish cutoff values for "high" versus "low" expression

  • Conduct inter-laboratory validation studies to ensure reproducibility

Previous research has established that lower expression of CSRNP3 is observed in high-risk patient groups compared to low-risk groups in certain diseases, with risk prediction models showing an AUC of 0.69 . This foundation can be built upon for further biomarker applications.

How can AI-based technologies be leveraged to develop improved antibodies against CSRNP3?

Recent advances in AI-based antibody design offer promising approaches for developing next-generation CSRNP3 antibodies:

• AI-Driven Epitope Prediction and Antibody Design:

  • Apply language models similar to IgLM to generate de novo CDRH3 sequences for CSRNP3 targeting

  • Use structural prediction tools like ImmuneBuilder to model antibody-antigen interactions

  • Design antibodies targeting specific functional domains of CSRNP3

  • This approach has shown success in generating diverse antibody candidates against targets like SARS-CoV-2

• Rational Design Based on Structural Information:

  • Generate structural models of CSRNP3 using AlphaFold or similar tools

  • Identify surface-exposed epitopes ideal for antibody recognition

  • Design antibodies with optimized complementarity-determining regions (CDRs)

  • Validate designs using molecular dynamics simulations

• High-Throughput Screening Integration:

  • Combine AI predictions with experimental screening approaches

  • Use technologies like Berkeley Lights Beacon platform for single-cell analysis

  • Screen AI-designed antibody candidates against CSRNP3-expressing cells

  • This approach has successfully identified antigen-specific heavy chain antibodies in other contexts

• Affinity Maturation Simulation:

  • Apply computational approaches to simulate affinity maturation processes

  • Generate in silico matured variants with potentially higher specificity and affinity

  • Experimentally validate top candidates

  • Focus on reducing cross-reactivity with other CSRNP family members

• Experimental Validation Pipeline:

  • Develop a standardized pipeline for testing AI-designed antibodies:

    • Express candidates as scFvs, Fabs, or full IgGs

    • Assess binding using surface plasmon resonance (SPR)

    • Evaluate specificity through Western blotting and immunofluorescence

    • Test function in relevant cellular assays

• Implementation Considerations:

  • Compare performance metrics between AI-designed and traditionally developed antibodies

  • Assess cost-efficiency and time-saving potential

  • Consider epitope coverage and diversity in the antibody portfolio

The integration of AI approaches with experimental validation has proven successful in generating diverse, high-quality antibodies against challenging targets and could significantly advance CSRNP3 research tools.

What are the cutting-edge approaches for studying CSRNP3 protein interactions in live cells?

To investigate CSRNP3 protein interactions with unprecedented spatial and temporal resolution:

• Advanced Live-Cell Imaging Techniques:

  • Implement FRET (Förster Resonance Energy Transfer) pairs:

    • Express CSRNP3 fused to a FRET donor (e.g., mTurquoise2)

    • Express potential interaction partners fused to acceptor fluorophores (e.g., mVenus)

    • Measure energy transfer as evidence of protein proximity

  • Apply FLIM (Fluorescence Lifetime Imaging Microscopy) to detect FRET with higher sensitivity

  • Use BiFC (Bimolecular Fluorescence Complementation) to visualize protein interactions directly

• Optogenetic Approaches:

  • Create optogenetic CSRNP3 fusion proteins to control activity with light

  • Combine with live imaging to observe downstream effects in real-time

  • Use optogenetic dimerization systems to induce or disrupt interactions on demand

• Proximity Labeling in Live Cells:

  • Express CSRNP3 fused to enzymes like:

    • TurboID or miniTurbo (engineered biotin ligases)

    • APEX2 (engineered ascorbate peroxidase)

  • These enzymes biotinylate proteins in close proximity to CSRNP3

  • Perform time-course experiments to capture dynamic interaction changes

  • Isolate biotinylated proteins and identify by mass spectrometry

• Super-Resolution Microscopy:

  • Apply techniques such as:

    • STORM (Stochastic Optical Reconstruction Microscopy)

    • PALM (Photoactivated Localization Microscopy)

    • SIM (Structured Illumination Microscopy)

  • Achieve resolution down to ~20nm to visualize CSRNP3 interactions at the nanoscale

  • Combine with multi-color imaging to track multiple interaction partners simultaneously

• Single-Molecule Tracking:

  • Label CSRNP3 with photoconvertible fluorescent proteins or quantum dots

  • Track individual molecules to reveal dynamics of:

    • Nuclear localization

    • Chromatin binding

    • Protein complex formation and dissociation

  • Analyze diffusion patterns to infer binding states

• CRISPR-Based Tagging:

  • Use CRISPR-Cas9 to insert fluorescent or affinity tags at the endogenous CSRNP3 locus

  • This maintains native expression levels and regulation

  • Combine with the imaging approaches above for physiologically relevant studies

• Correlative Light and Electron Microscopy (CLEM):

  • Identify CSRNP3-containing complexes by fluorescence microscopy

  • Examine the same structures at ultrastructural resolution with electron microscopy

  • This provides context for interactions within nuclear architecture

These cutting-edge approaches overcome limitations of traditional biochemical methods by allowing the study of CSRNP3 interactions in their native cellular environment with high spatial and temporal resolution.

How might CSRNP3's role differ across various tissue and disease contexts based on current research findings?

Emerging evidence suggests CSRNP3 may have context-dependent roles that vary significantly across tissues and disease states:

By systematically investigating these context-dependent roles, researchers can develop a more nuanced understanding of CSRNP3's biology and its potential as a biomarker or therapeutic target across different pathological conditions.

What are the most promising future directions for CSRNP3 antibody applications in research?

Several promising research directions emerge from current understanding of CSRNP3 biology and antibody technology:

• Development of Multimodal Imaging Probes:

  • Create antibody-based imaging agents that combine:

    • FITC or other fluorophores for optical imaging

    • Radiolabels for PET/SPECT imaging

    • MRI contrast agents

  • These would enable translation between microscopic and whole-body imaging applications

  • Particularly valuable for tracking CSRNP3 expression in disease models

• Therapeutic Target Validation:

  • Use CSRNP3 antibodies to validate it as a potential therapeutic target

  • Develop blocking antibodies that interfere with specific CSRNP3 functions

  • Assess the impact of CSRNP3 inhibition on disease progression in relevant models

  • Focus on contexts where CSRNP3 shows strong prognostic associations

• Single-Cell Analysis Applications:

  • Adapt CSRNP3 antibodies for single-cell technologies:

    • Mass cytometry (CyTOF) using metal-tagged antibodies

    • CITE-seq for simultaneous protein and RNA profiling

    • Spatial transcriptomics with protein co-detection

  • These approaches would reveal heterogeneity in CSRNP3 expression and function at unprecedented resolution

• Antibody Fragment Development:

  • Engineer smaller antibody formats (Fabs, scFvs, nanobodies) against CSRNP3

  • These may offer improved tissue penetration and reduced background

  • Apply strategies similar to those used for other targets, such as the VHH-rabbit IgG heavy chain antibody approach

• Spatial Biology Integration:

  • Incorporate CSRNP3 antibodies into multiplexed spatial profiling platforms

  • Map CSRNP3 expression in relation to tissue architecture and cellular neighborhoods

  • Correlate with immune infiltrates and other microenvironmental features

• Cross-Platform Standardization:

  • Develop reference standards for CSRNP3 quantification across platforms

  • Enable reliable comparison of results between technologies and laboratories

  • Critical for clinical biomarker development

• AI-Integrated Antibody Design and Analysis:

  • Combine AI approaches for antibody design with automated image analysis

  • Create feedback loops where imaging results inform next-generation antibody designs

  • This integrative approach could accelerate discovery of optimal CSRNP3-targeting tools