LRRC3C Antibody, FITC conjugated

Basic Research Considerations

• What is LRRC3C and what is its significance in cellular signaling?

  LRRC3C (Leucine Rich Repeat Containing 3C) is a protein-coding gene that plays a role in multiple cellular pathways. Research indicates that LRRC3C functions primarily in the cytoplasm and is involved in cytokine-mediated signaling pathways . It demonstrates protein kinase inhibitor activity and participates in the negative regulation of the JAK-STAT cascade and protein kinase activity . An important paralog of this gene is LRRC3B .

  The protein contains leucine-rich repeat domains which are common structural motifs that form amphipathic surfaces mediating protein-protein interactions. In research contexts, LRRC3C's regulatory role in JAK-STAT signaling makes it relevant for studies investigating immune response mechanisms and cellular signaling perturbations in disease states.

• What is the molecular characterization of commercially available LRRC3C antibodies?

  LRRC3C antibodies are typically generated from rabbits immunized with synthetic peptides or recombinant proteins representing specific regions of the human LRRC3C protein. Available research-grade antibodies target different epitopes:

  Antibody Type	Target Epitope	Host	Applications	Purification Method
  Polyclonal (ABIN1910578)	AA 234-260 (C-terminal)	Rabbit	WB, ELISA	Affinity purified
  Polyclonal	AA 42-224	Rabbit	ELISA, IHC	Affinity purified
  Polyclonal	AA 42-275	Rabbit	WB, IHC, ICC	Not specified

  The antibodies are generated against KLH-conjugated synthetic peptides or recombinant human LRRC3C protein fragments . Specificity is typically confirmed through testing against human samples, with most antibodies showing reactivity specifically against human LRRC3C.

• How does FITC conjugation affect antibody functionality and what are its spectral properties?

  FITC (Fluorescein Isothiocyanate) conjugation creates a covalent bond between the dye and antibody through reaction with primary amines, primarily at lysine residues. This conjugation:

  • Enables direct visualization without secondary antibodies

  • Provides fluorescence with excitation maximum at ~495nm and emission at ~525nm

  • Produces a bright yellow-green fluorescence signal

  While conjugation is designed to preserve the biological activity of the antibody, researchers should verify that FITC labeling doesn't interfere with epitope recognition in their specific experimental system . FITC conjugation is particularly valuable for applications requiring direct detection such as flow cytometry, immunofluorescence microscopy, and FACS analysis.

  The quantum yield and photostability of FITC make it suitable for most standard fluorescence applications, though it is somewhat susceptible to photobleaching compared to newer generation fluorophores .

Experimental Methodology

• What are the optimal protocols for using LRRC3C Antibody, FITC conjugated in immunofluorescence studies?

  When conducting immunofluorescence with FITC-conjugated LRRC3C antibodies, researchers should follow these methodological considerations:

  1. Sample Preparation:

     • Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

     • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  2. Blocking and Antibody Application:

     • Block with PBS containing 10% fetal bovine serum for 20 minutes at room temperature

     • Dilute FITC-conjugated LRRC3C antibody 1:500 in PBS/10% FBS

     • Incubate for 1 hour at room temperature in the dark

  3. Post-Incubation Processing:

     • Wash cells 2 × 5 minutes with PBS

     • Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

  4. Visualization:

     • Observe using a fluorescence microscope equipped with a FITC filter (excitation ~495nm, emission ~525nm)

     • Minimize exposure time to prevent photobleaching

  Critical considerations include protecting the antibody from light exposure during all steps, as continuous exposure will cause gradual loss of fluorescence . Additionally, empirical determination of the optimal antibody dilution may be necessary depending on your specific cell line or tissue type.

• What controls should be implemented when using LRRC3C Antibody, FITC conjugated in flow cytometry?

  Proper experimental controls are critical for accurate flow cytometry analysis with FITC-conjugated LRRC3C antibodies:

  Required Controls:

  1. Unstained Control:

     • Cells processed identically but without any antibody

     • Establishes autofluorescence baseline and assists in gating strategy

  2. Isotype Control:

     • FITC-conjugated rabbit IgG of the same isotype at identical concentration

     • Controls for non-specific binding of immunoglobulins

  3. FMO (Fluorescence Minus One) Control:

     • In multicolor panels, include all fluorophores except FITC

     • Helps establish proper compensation settings

  4. Antibody Pre-absorption Control:

     • Pre-incubate antibody with excess LRRC3C peptide before cell staining

     • Confirms specificity of the signal

  5. Positive Control:

     • Cells known to express LRRC3C (based on literature)

     • Validates antibody performance

  The specificity can be further confirmed by observing quenching of fluorescence upon pre-incubation with anti-FITC antibodies, as demonstrated in flow cytometry validation studies showing significant signal reduction when FITC antibodies are pre-incubated with the sample .

• How should researchers optimize Western Blot protocols when using LRRC3C Antibody, FITC conjugated?

  Western blotting with FITC-conjugated antibodies requires specific protocol adaptations:

  1. Sample Preparation:

     • Standard protein extraction and denaturation protocols are suitable

     • Load 20-50 μg of protein per lane depending on expression level

  2. Transfer and Blocking:

     • Transfer proteins to PVDF membrane (preferred over nitrocellulose for fluorescence detection)

     • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  3. Antibody Incubation:

     • Dilute LRRC3C Antibody, FITC conjugated at 1:500 to 1:1000 in blocking buffer

     • Incubate overnight at 4°C in a light-protected container

  4. Detection Optimization:

     • Wash extensively (4-5 times, 5 minutes each) to reduce background

     • Visualize using a fluorescence imager with appropriate filters

     • Protect membrane from light during all steps post-antibody incubation

  5. Signal Enhancement (if needed):

     • For weak signals, consider using anti-FITC secondary antibodies

     • Anti-FITC antibodies can amplify signal while maintaining specificity

  The LRRC3C antibody has been successfully validated for WB applications detecting the appropriate molecular weight bands corresponding to human LRRC3C protein .

Advanced Research Applications

• What are the methodological considerations for using LRRC3C Antibody, FITC conjugated in multiplex immunofluorescence studies?

  Multiplex immunofluorescence with FITC-conjugated LRRC3C antibodies requires careful experimental design:

  1. Spectral Considerations:

     • FITC emits at ~525nm (green), so select compatible fluorophores that minimize spectral overlap

     • Appropriate combinations include: FITC + TRITC/Cy3 + Cy5 + DAPI

  2. Panel Design Strategy:

     • Assign FITC to antigens with moderate to high expression levels

     • Reserve brighter fluorophores (e.g., Alexa Fluors) for lower-abundance targets

  3. Sequential Staining Protocol:

     • For co-localization studies with other rabbit antibodies, use sequential staining approaches

     • Apply LRRC3C-FITC first, fix with 4% PFA briefly, then apply additional antibodies

  4. Image Acquisition Optimization:

     • Capture FITC channel first to minimize photobleaching

     • Use narrow bandpass filters to minimize bleed-through

     • Perform sequential scanning rather than simultaneous acquisition

  5. Cross-Reactivity Prevention:

     • When using multiple rabbit-derived antibodies, employ Fab fragment blocking

     • Consider tyramide signal amplification (TSA) for sequential multiplexing

  Research examining Fc-engineered antibodies in similar experimental designs demonstrated that capturing detailed cellular responses requires proper multiplex design to delineate cell type-specific effects . Studies have successfully employed FITC-labeled antibodies in combination with other fluorophores to characterize complex cellular interactions in immunological contexts .

• How can researchers interpret discrepancies between LRRC3C protein detection via FITC-antibodies versus mRNA expression data?

  Discrepancies between protein and mRNA levels are common in biological research and require systematic analysis:

  1. Biological Explanations:

     • Post-transcriptional regulation (miRNAs, RNA-binding proteins)

     • Post-translational modifications affecting antibody epitope recognition

     • Protein turnover rates differing from mRNA stability

     • Alternative splicing creating protein isoforms that may not contain the antibody epitope

  2. Technical Validation Approaches:

     • Verify antibody specificity using knockout/knockdown systems

     • Test multiple antibodies targeting different LRRC3C epitopes

     • Perform immunoprecipitation followed by mass spectrometry

     • Use quantitative western blotting with recombinant protein standards

  3. Integrated Analysis Strategy:

     • Correlate findings with functional assays of LRRC3C-associated pathways

     • Examine protein localization data for insight into functional activity

     • Consider temporal dynamics of expression (mRNA vs protein lag time)

  Recent immunological research demonstrates that protein expression determined by antibody-based methods can reveal functional aspects not evident in transcriptomic data alone . For LRRC3C specifically, its role in protein kinase inhibition and JAK-STAT pathway regulation suggests that post-translational modifications may significantly impact its detection and function beyond mRNA expression levels .

• What role does LRRC3C play in cytokine-mediated signaling and immune response regulation?

  LRRC3C has been characterized as a regulatory component in immune signaling pathways:

  1. JAK-STAT Pathway Regulation:

     • LRRC3C exhibits protein kinase inhibitor activity

     • It functions in the negative regulation of the JAK-STAT cascade

     • This pathway is critical for cytokine signaling and immune response coordination

  2. Potential Immunomodulatory Functions:

     • The negative regulation of protein kinase activity suggests a role in dampening signaling responses

     • May function as a checkpoint in preventing aberrant immune activation

  3. Related Research Context:

     • Similar leucine-rich repeat containing proteins have been implicated in:

       • Pattern recognition receptor function

       • Protein-protein interactions in immune signaling complexes

       • Regulation of inflammatory responses

  4. Experimental Approaches to Study Function:

     • Co-immunoprecipitation to identify interaction partners

     • Phosphorylation studies to examine effects on JAK/STAT activation

     • Cytokine stimulation assays with LRRC3C knockdown/overexpression

  While direct research on LRRC3C in cytokine signaling is still developing, studies on related immune pathways have employed antibody-based approaches to characterize key regulatory mechanisms . The advancement of Fc-engineered antibodies demonstrates the importance of understanding protein-level regulation in immune signaling cascades, which may have parallels to LRRC3C function .

Troubleshooting and Quality Control

• What are common causes of high background when using LRRC3C Antibody, FITC conjugated, and how can they be addressed?

  High background is a common challenge with FITC-conjugated antibodies that can be systematically addressed:

  Problem Source	Manifestation	Solution
  Insufficient blocking	Diffuse background across all cells/tissues	Increase blocking time to 1-2 hours; try different blocking agents (BSA, normal serum, casein)
  Excessive antibody concentration	Strong non-specific signal	Perform antibody titration; optimal dilution typically 1:500 for LRRC3C-FITC
  Photobleaching and autofluorescence	Tissue-specific background	Use freshly prepared samples; include antifade reagents; use spectral unmixing
  FITC photosensitivity	Signal fading during imaging	Protect from light; minimize exposure time; capture FITC channel first
  Inadequate washing	General haziness	Extend wash steps (minimum 3 washes, 5 minutes each) with gentle agitation

  Additionally, FITC is particularly sensitive to pH changes, with optimal fluorescence at pH 8.0-9.0. Buffer systems should be carefully controlled, and mounting media should be specifically formulated for fluorescence preservation .

  If high background persists despite these measures, consider:

  • Pre-absorbing the antibody with cells/tissues lacking the target

  • Using secondary amplification with anti-FITC antibodies for more controlled detection

  • Employing alternative detection systems (HRP-conjugated anti-FITC with TSA amplification)

• How can researchers verify the specificity of LRRC3C Antibody, FITC conjugated for their experimental system?

  Verifying antibody specificity is crucial for meaningful experimental results:

  1. Peptide Competition Assay:

     • Pre-incubate antibody with excess LRRC3C peptide (the immunogen)

     • Compare staining with and without peptide blocking

     • Specific signal should be significantly reduced after peptide competition

  2. Genetic Validation:

     • Test antibody in LRRC3C knockout/knockdown systems

     • Use CRISPR-Cas9 edited cell lines lacking LRRC3C

     • Compare with wild-type cells showing normal expression

  3. Cross-Validation With Alternative Methods:

     • Compare protein detection with mRNA expression (qPCR, RNA-seq)

     • Use alternative antibodies targeting different LRRC3C epitopes

     • Complement with mass spectrometry-based protein identification

  4. Technical Controls:

     • Isotype control at equivalent concentration to test non-specific binding

     • Secondary-only controls (for indirect detection methods)

     • Unstained controls to establish autofluorescence baseline

  Research protocols indicate that validation strategies should be tailored to the specific application. For flow cytometry, demonstrating fluorescence quenching upon anti-FITC antibody pre-incubation provides a reliable specificity check . For immunohistochemistry, parallel staining with multiple LRRC3C antibodies targeting different epitopes is recommended.

• What approaches can mitigate photobleaching when working with FITC-conjugated LRRC3C antibodies?

  FITC is known to be relatively susceptible to photobleaching, which can be managed with several techniques:

  1. Sample Preparation Optimization:

     • Use fresh reagents and prepare samples immediately before imaging

     • Store prepared slides/plates in light-protected containers at 4°C

     • Consider using alternative mounting media with anti-fade properties

  2. Imaging Protocol Adjustments:

     • Minimize exposure time and illumination intensity

     • Use neutral density filters to reduce excitation light intensity

     • Capture FITC channels first in multi-channel imaging workflows

     • Employ interval shuttering during extended imaging sessions

  3. Chemical Photoprotection:

     • Include anti-fade reagents in mounting media (e.g., n-propyl gallate, DABCO)

     • Use oxygen scavenger systems for live-cell imaging (glucose oxidase/catalase)

     • Consider commercial anti-fade mounting media specifically designed for FITC

  4. Alternative Approaches:

     • For critical experiments, consider using more photostable alternatives like Alexa Fluor 488

     • Employ signal amplification systems (tyramide signal amplification)

     • Use computational methods to correct for photobleaching in time-series experiments

  Studies emphasize the importance of protecting FITC-conjugated antibodies from light during all experimental steps, as continuous exposure will cause gradual loss of fluorescence . When performing extended imaging sessions, researchers should consider capturing reference images at regular intervals to quantify and compensate for photobleaching effects.

Advanced Research Directions

• How do LRRC3C expression patterns correlate with immune cell activation states in disease models?

  Understanding LRRC3C expression in immune contexts requires integrated experimental approaches:

  1. Cellular Distribution Analysis:

     • Flow cytometry with LRRC3C-FITC antibodies enables quantification across immune subsets

     • Single-cell resolution studies can reveal heterogeneity within populations

     • Expression changes during activation can be tracked with time-course experiments

  2. Functional Correlation Studies:

     • Parallel analysis of LRRC3C expression with activation markers (CD69, CD25, etc.)

     • Cytokine production profiles correlated with LRRC3C levels

     • Phospho-flow approaches to examine JAK-STAT pathway activity

  3. Disease-Specific Considerations:

     • In inflammatory conditions, examine correlation with pro-inflammatory cytokine levels

     • In cancer models, analyze expression in tumor-infiltrating lymphocytes

     • In infection models, monitor changes during pathogen clearance

  Recent immunological research demonstrates that antibody-based approaches can reveal crucial insights into cellular activation states beyond what is evident from genomic analyses alone . The potential role of LRRC3C in negative regulation of JAK-STAT signaling suggests it may function as a checkpoint in immune activation, making its expression patterns particularly relevant in understanding disease immunopathology .

• What are the latest methodological advances in using fluorescently-labeled antibodies for studying protein-protein interactions involving LRRC3C?

  Advanced fluorescence techniques offer powerful approaches for studying LRRC3C interactions:

  1. Proximity Ligation Assay (PLA):

     • Uses FITC-conjugated antibodies with complementary probes

     • Generates fluorescent signal only when proteins are within 40nm

     • Can detect endogenous protein interactions without overexpression

  2. Förster Resonance Energy Transfer (FRET):

     • Combines FITC-labeled LRRC3C antibodies with compatible acceptor fluorophores

     • Measures energy transfer that occurs only at molecular distances (1-10nm)

     • Provides quantitative measure of interaction strength

  3. Fluorescence Correlation Spectroscopy (FCS):

     • Analyzes diffusion characteristics of FITC-labeled complexes

     • Can detect changes in molecular size upon complex formation

     • Works with endogenous proteins at physiological concentrations

  4. Advanced Microscopy Platforms:

     • Super-resolution techniques overcome diffraction limit for nanoscale localization

     • Light sheet microscopy enables whole-cell or tissue-level interaction mapping

     • Live-cell imaging captures dynamic interaction events

  Innovative approaches in immunological research have demonstrated that fluorescently-labeled antibodies can be engineered for enhanced specificity and functionality in complex cellular contexts . These methodologies provide opportunities to decipher LRRC3C's role in protein-protein interaction networks that regulate cytokine signaling and JAK-STAT pathway modulation .

• How can researchers integrate LRRC3C protein expression data with other -omics datasets for systems biology approaches?

  Integrative analysis of LRRC3C requires sophisticated data integration strategies:

  1. Multi-omics Data Collection:

     • Protein expression (antibody-based): FITC-labeled LRRC3C quantification via flow cytometry or microscopy

     • Transcriptomics: RNA-seq for mRNA expression

     • Phosphoproteomics: MS-based detection of signaling pathway activities

     • Interactomics: Protein-protein interaction networks via IP-MS

  2. Computational Integration Methods:

     • Correlation analysis across data types

     • Network reconstruction algorithms to place LRRC3C in functional modules

     • Machine learning approaches to identify patterns across datasets

     • Causal inference models to determine regulatory relationships

  3. Functional Validation Experiments:

     • Targeted perturbation of LRRC3C (CRISPR, RNAi)

     • Measurement of effects across multiple molecular layers

     • Time-series experiments to capture dynamic responses

  4. Visualization and Analysis Platforms:

     • Pathway mapping tools to contextualize LRRC3C in signaling networks

     • Interactive visualization platforms for complex data exploration

     • Statistical frameworks for integrating heterogeneous data types

  Recent advances in single-cell multi-omics approaches are particularly relevant, as they allow correlation of LRRC3C protein expression with transcriptional states at single-cell resolution. Research on immune signaling pathways has demonstrated that integrating antibody-based protein detection with transcriptional profiling can reveal functional insights that would be missed by either approach alone .