REG4 Antibody, FITC conjugated

What is REG4 and why is it an important research target?

REG4 (Regenerating islet-derived protein 4) is a calcium-independent lectin that displays mannose-binding specificity and maintains carbohydrate recognition activity in acidic environments. The protein is primarily involved in inflammatory and metaplastic responses of the gastrointestinal epithelium . As a secreted protein associated with gastrointestinal tumors, REG4 represents an important biomarker for various cancers and inflammatory conditions of the digestive system. REG4 expression analysis using specific antibodies enables researchers to investigate its role in disease pathogenesis and potential utility as a diagnostic marker.

What are the key specifications of commercially available FITC-conjugated REG4 antibodies?

Commercial FITC-conjugated REG4 antibodies, such as the rabbit polyclonal antibody described in the literature, typically target specific amino acid sequences of human REG4 (e.g., AA 29-140) . These antibodies are characterized by the following specifications:

The high purity and specific targeting make these antibodies suitable for precise detection of REG4 in research applications .

What is the principle behind FITC conjugation, and how does it benefit REG4 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation involves the covalent attachment of the FITC fluorophore to antibody molecules, typically via primary amine groups on lysine residues. This chemical modification enables direct visualization of antibody binding through fluorescence detection without requiring secondary antibodies. The FITC fluorophore has an excitation maximum at approximately 495 nm and emission maximum around 519 nm, producing a bright green fluorescence when excited with the appropriate wavelength.

For REG4 antibody applications, FITC conjugation provides several methodological advantages:

• Direct detection in flow cytometry and fluorescence microscopy

• Reduction of background from secondary antibody binding

• Compatibility with multicolor analysis when combined with other fluorophores

• Stable signal for quantitative measurements

• Simplified protocols with fewer incubation and washing steps

These benefits make FITC-conjugated REG4 antibodies particularly valuable for investigating REG4 expression in complex tissue samples and cell populations .

How should FITC-conjugated REG4 antibodies be incorporated into immunofluorescence microscopy protocols?

For optimal immunofluorescence microscopy using FITC-conjugated REG4 antibodies, follow this methodological approach:

• Sample preparation:

  • Fix cells or tissue sections with 4% paraformaldehyde (10-15 minutes)

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 minutes) if intracellular staining is required

  • Block with 1-5% BSA or serum in PBS (30-60 minutes)

• Antibody incubation:

  • Dilute FITC-conjugated REG4 antibody to optimal concentration (typically starting at 1:100-1:500 dilution)

  • Incubate samples with diluted antibody in blocking buffer for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber protected from light

  • Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/mL, 5 minutes)

  • Mount with anti-fade mounting medium

  • Seal coverslip edges with nail polish for long-term storage

• Controls to include:

  • Negative control: isotype-matched FITC-conjugated IgG at the same concentration

  • Positive control: tissue or cell line known to express REG4

  • Autofluorescence control: unstained sample

The recommended antibody concentration should be determined empirically for each application, but generally ranges between 0.5-5 μg/mL .

What are the optimized protocols for using FITC-conjugated REG4 antibodies in flow cytometry?

For flow cytometric analysis using FITC-conjugated REG4 antibodies, the following detailed protocol is recommended:

• Cell preparation:

  • Harvest cells and wash in cold PBS containing 0.1% sodium azide

  • For intracellular staining, fix cells with 4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS

• Antibody staining:

  • Resuspend cells at 1 × 10^6 cells/100 μL in staining buffer (PBS, 0.5% BSA, 0.1% sodium azide)

  • Add FITC-conjugated REG4 antibody at ≤ 0.5 μg per million cells or at manufacturer's recommended dilution

  • Incubate for 30-45 minutes at 4°C in the dark

  • Wash twice with staining buffer

• Instrument setup:

  • Calibrate flow cytometer with appropriate FITC-positive and negative controls

  • Adjust voltage settings to place negative population in first decade of fluorescence histogram

  • Acquire at least 10,000 events per sample for statistical significance

• Analysis considerations:

  • Gate on viable cells using forward/side scatter or viability dye

  • Compare to isotype control to determine positive staining threshold

  • For quantification, calculate percent positive cells or mean fluorescence intensity (MFI)

For demonstrating specificity, consider pre-blocking experiments with unlabeled REG4 antibody before staining with the FITC-conjugated antibody .

How do Western blotting protocols need to be adapted for FITC-conjugated REG4 antibodies?

While FITC-conjugated antibodies are not typically the first choice for Western blotting, they can be used with specific protocol modifications:

• Sample preparation and gel electrophoresis:

  • Prepare protein samples in standard reducing buffer

  • Run SDS-PAGE as normal

  • Transfer proteins to PVDF or nitrocellulose membrane

• Membrane processing:

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

  • Incubate with FITC-conjugated REG4 antibody (1:500-1:1000) in blocking buffer overnight at 4°C in the dark

  • Wash extensively with TBST (5-6 times, 5 minutes each)

• Detection:

  • Visualize directly using a fluorescence scanner or imager with appropriate excitation (488 nm) and emission (515-530 nm) filters

  • For documentation, capture images using a CCD camera system with FITC filter sets

  • Protect membrane from light throughout the procedure to prevent photobleaching

• Important considerations:

  • FITC conjugation may reduce antibody sensitivity compared to unconjugated antibodies

  • Expected molecular weight of human REG4 is approximately 17 kDa

  • Include positive control lysates from cells known to express REG4

  • For quantification, use fluorescence intensity analysis software

For optimal results in Western blotting applications, unconjugated REG4 antibodies with enzyme-labeled secondary antibodies may provide better sensitivity than direct FITC conjugates .

How can FITC-conjugated REG4 antibodies be used for multiplex analysis with other biomarkers?

For multiplex analysis combining FITC-conjugated REG4 antibodies with other biomarkers:

• Fluorophore selection strategy:

  • Choose fluorophores with minimal spectral overlap with FITC (excitation 495nm/emission 519nm)

  • Recommended combinations: FITC (REG4) + PE (red, 565nm) + APC (far-red, 660nm)

  • For 4+ color experiments, consider: FITC + PE + PE-Cy5 + APC or APC-Cy7

• Multiplexed immunofluorescence microscopy protocol:

  • Perform sequential staining for each primary antibody-fluorophore pair

  • Include blocking steps between antibodies from the same species

  • Use spectral unmixing for fluorophores with partial overlap

  • Apply nuclear counterstain (DAPI) as final step

• Multiplex flow cytometry considerations:

  • Perform single-color compensation controls for each fluorophore

  • Use FMO (Fluorescence Minus One) controls to set proper gates

  • Consider viability dyes (e.g., 7-AAD or far-red dyes) to exclude dead cells

• Potential biomarker combinations with REG4:

  • Epithelial markers: E-cadherin, EpCAM

  • Cancer stem cell markers: CD133, CD44, LGR5

  • Proliferation markers: Ki-67

  • Lineage markers: CDX2, MUC2

This approach enables simultaneous assessment of REG4 expression alongside other relevant biomarkers in complex tissues or heterogeneous cell populations, providing insight into REG4's relationship with cellular phenotypes and states .

What approaches can resolve antibody cross-reactivity issues when working with FITC-conjugated REG4 antibodies?

When encountering potential cross-reactivity with FITC-conjugated REG4 antibodies, implement these advanced troubleshooting strategies:

• Experimental validation of specificity:

  • Peptide competition assay: Pre-incubate antibody with excess recombinant REG4 protein (5-10 μg/mL) before staining

  • Knockout/knockdown controls: Compare staining in REG4-knockout cells (CRISPR-Cas9) or REG4-silenced cells (siRNA)

  • Orthogonal detection methods: Validate with alternative REG4 antibody clones or RNA expression data

• Optimization of staining conditions:

  • Titrate antibody concentration to minimize background (typically 0.1-1 μg/mL)

  • Modify blocking reagents (try 5% BSA, normal serum, or commercial blockers)

  • Adjust incubation temperature and duration (4°C vs. room temperature, 1-hour vs. overnight)

  • Include 0.1% Tween-20 in wash buffers to reduce non-specific binding

• Advanced controls:

  • Multi-epitope approach: Use antibodies targeting different REG4 epitopes

  • Species-specific isotype controls at matching concentrations

  • Pre-adsorption with related proteins (other REG family members)

• Data analysis approaches:

  • Quantify signal-to-background ratio under different conditions

  • Apply spectral unmixing algorithms to separate autofluorescence

  • Use ratiometric analysis against housekeeping proteins

For particularly challenging samples, consider alternative detection methods such as RNAscope for mRNA detection to complement protein analysis .

How can researchers quantitatively assess REG4 expression levels using FITC-conjugated antibodies?

Quantitative assessment of REG4 expression using FITC-conjugated antibodies requires rigorous methodological approaches:

• Flow cytometry quantification:

  • Use calibration beads with defined FITC molecules (MESF - Molecules of Equivalent Soluble Fluorochrome)

  • Create standard curve of mean fluorescence intensity (MFI) vs. known MESF values

  • Convert sample MFI to approximate antibody binding capacity

  • Express as relative fluorescence units (RFU) or molecules per cell

• Quantitative immunofluorescence microscopy:

  • Include calibration slides with known fluorophore concentrations

  • Maintain identical acquisition parameters across all samples

  • Measure integrated density or mean gray value of defined regions of interest

  • Subtract background from areas without specific staining

  • Normalize to cell number or tissue area

• Standardization considerations:

  • Use internal controls (constitutively expressed proteins) for normalization

  • Include reference cell lines with known REG4 expression levels

  • Account for fluorophore:protein ratio variability between antibody lots

  • Verify linear dynamic range of detection system

• Quantitative data analysis:

  • For flow cytometry: Compare percent positive cells and MFI values

  • For microscopy: Use automated image analysis software for unbiased quantification

  • Apply appropriate statistical tests based on data distribution

  • Consider cell-to-cell variability through single-cell analysis approaches

This quantitative approach allows for meaningful comparisons of REG4 expression between different experimental conditions, cell types, or patient samples .

What are the current challenges and solutions in using FITC-conjugated REG4 antibodies for detecting post-translational modifications?

Detecting post-translational modifications (PTMs) of REG4 using FITC-conjugated antibodies presents several methodological challenges:

• Current challenges:

  • Limited availability of modification-specific REG4 antibodies

  • Potential epitope masking when PTMs occur near antibody binding sites

  • Competition between modified and unmodified forms for antibody binding

  • Sensitivity limitations for low-abundance modified forms

  • FITC photobleaching during extended imaging protocols

• Methodological solutions:

  • Combined immunoprecipitation strategy:

    1. Immunoprecipitate total REG4 with unconjugated antibody

    2. Probe immunoprecipitated material with FITC-conjugated PTM-specific antibodies (e.g., anti-phosphotyrosine-FITC)

    3. Verify with mass spectrometry for precise PTM identification

  • Sequential epitope exposure technique:

    1. Perform mild antigen retrieval optimized for the specific PTM

    2. Use epitope-specific REG4 antibody alongside PTM-specific antibodies

    3. Apply spectral unmixing for multiplexed PTM detection

• Advanced detection approaches:

  • Proximity ligation assay (PLA) to confirm colocalization of REG4 and specific PTMs

  • FRET-based assays to detect conformational changes associated with REG4 modification

  • Single-molecule microscopy for detecting rare modification events

• Emerging techniques:

  • Development of site-specific antibodies for known REG4 modifications

  • Genetic incorporation of unnatural amino acids at potential modification sites

  • Application of super-resolution microscopy for spatial distribution of modified REG4

These approaches help overcome the limitations of standard techniques and provide deeper insights into how post-translational modifications regulate REG4 function in physiological and pathological contexts .

What are the most common technical issues encountered with FITC-conjugated REG4 antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with FITC-conjugated REG4 antibodies. Here are the most common issues and their methodological solutions:

• High background fluorescence:

  • Cause: Non-specific binding, autofluorescence, or excessive antibody concentration

  • Solutions:

    • Increase blocking time (1-2 hours) with 5% BSA or 10% serum

    • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

    • Titrate antibody to optimal concentration (typically 0.1-0.5 μg/million cells)

    • Include 0.05% Tween-20 in wash buffers

    • Use Sudan Black B (0.1-0.3%) to quench tissue autofluorescence

• Weak or absent signal:

  • Cause: Insufficient antibody concentration, epitope masking, or photobleaching

  • Solutions:

    • Optimize fixation time (overfixation can mask epitopes)

    • Try heat-induced epitope retrieval (citrate buffer, pH 6.0)

    • Increase antibody concentration or incubation time

    • Use signal amplification systems (e.g., TSA)

    • Protect from light during all steps

    • Check for proper storage conditions (-20°C, avoid freeze-thaw cycles)

• Inconsistent staining patterns:

  • Cause: Heterogeneous sample preparation or antibody degradation

  • Solutions:

    • Standardize fixation and permeabilization protocols

    • Aliquot antibody upon receipt to minimize freeze-thaw cycles

    • Include positive control samples in each experiment

    • Prepare fresh dilutions for each experiment

    • Verify antibody integrity with dot blot before use

• Cross-reactivity:

  • Cause: Antibody binding to proteins with similar epitopes

  • Solutions:

    • Validate with REG4-knockout or knockdown controls

    • Perform peptide competition assays

    • Use higher dilutions to favor high-affinity specific binding

    • Employ more stringent washing protocols

A systematic approach to troubleshooting that evaluates each step of the protocol will help identify and resolve most technical issues with FITC-conjugated REG4 antibodies .

How should researchers validate and quality control FITC-conjugated REG4 antibodies before experimental use?

A comprehensive validation workflow for FITC-conjugated REG4 antibodies ensures reliable experimental results:

• Initial functional assessment:

  • Spectral analysis:

    • Measure absorbance (peak at ~495 nm) and emission spectra (peak at ~519 nm)

    • Calculate fluorophore:protein ratio (typically 3-6 FITC molecules per antibody)

    • Assess for signs of aggregation (abnormal peak broadening)

  • SDS-PAGE evaluation:

    • Run reduced sample to confirm expected molecular weight (~150 kDa)

    • Visualize directly with fluorescence scanner before protein staining

    • Check for degradation products or aggregates

• Specificity validation:

  • Positive control testing:

    • Stain REG4-expressing cell lines (e.g., certain colorectal cancer lines)

    • Verify signal in tissues known to express REG4 (e.g., intestinal tissues)

  • Negative control testing:

    • Use REG4-negative cell lines or tissues

    • Apply isotype-matched FITC-conjugated control antibody

    • Perform knockdown/knockout validation when possible

• Performance assessment:

  • Titration analysis:

    • Test serial dilutions to determine optimal signal-to-noise ratio

    • Plot titration curve (concentration vs. signal intensity)

    • Determine minimum effective concentration

  • Reproducibility testing:

    • Compare lot-to-lot variation using standardized samples

    • Assess performance across different users and instruments

    • Evaluate stability after multiple freeze-thaw cycles

• Documentation and quality control record:

  Parameter	Acceptance Criteria	Test Result
  Spectral properties	Ex: 495±5nm, Em: 519±5nm	[Result]
  F/P ratio	3-6 FITC/antibody	[Result]
  MW confirmation	~150 kDa (main band)	[Result]
  Signal in positive control	≥3x background	[Result]
  Signal in negative control	≤1.5x background	[Result]
  Optimal working dilution	[Determined value]	[Result]
  Lot-to-lot consistency	CV < 15%	[Result]

This systematic approach ensures that FITC-conjugated REG4 antibodies meet the necessary quality standards for reliable experimental use .

How can FITC-conjugated REG4 antibodies be applied in high-throughput screening applications?

FITC-conjugated REG4 antibodies offer several advantages for high-throughput screening (HTS) applications:

• Automated microscopy platforms:

  • Methodology:

    • Seed cells in 96- or 384-well optical-bottom plates

    • Perform automated fixation and staining using liquid handling systems

    • Acquire images using high-content screening microscopes with FITC filter sets

    • Implement automated image analysis algorithms for quantification

  • Applications:

    • Screen compound libraries for modulators of REG4 expression

    • Evaluate genetic knockdown/overexpression effects on REG4 levels

    • Assess drug combinations for synergistic effects on REG4 regulation

• Flow cytometry-based HTS:

  • Protocol optimization:

    • Miniaturize staining protocols to 96-well format

    • Reduce volumes to 20-50 μL per well

    • Implement automated sample handling and acquisition

    • Develop gating strategies for consistent analysis

  • Applications:

    • Profile REG4 expression across cell line panels

    • Screen for antibodies that block REG4-ligand interactions

    • Monitor responses to treatment conditions in real-time

• Multiplexed bead-based assays:

  • Methodology:

    • Couple anti-REG4 capture antibodies to coded microbeads

    • Detect bound REG4 with FITC-conjugated detection antibodies

    • Analyze using flow cytometers with bead discrimination capabilities

  • Advantages:

    • Simultaneous analysis of multiple analytes

    • Reduced sample volume requirements

    • Improved throughput compared to traditional ELISA

• Quantitative considerations:

  • Include standard curves using recombinant REG4 protein

  • Implement robust statistical analysis (Z'-factor >0.5 for assay validation)

  • Develop machine learning algorithms for complex phenotypic profiling

These high-throughput approaches enable large-scale studies of REG4 biology, drug discovery efforts, and biomarker validation across numerous samples simultaneously .

What are the recent advances in combining FITC-conjugated REG4 antibodies with other emerging technologies?

Recent technological developments have expanded the applications of FITC-conjugated REG4 antibodies:

• Integration with single-cell technologies:

  • CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

    • Conjugate REG4 antibodies with both FITC and oligonucleotide barcodes

    • Simultaneously profile REG4 protein expression and whole-transcriptome RNA

    • Correlate REG4 protein levels with gene expression signatures at single-cell resolution

  • Mass cytometry adaptation:

    • Use metal-tagged secondary antibodies against FITC

    • Enable highly multiplexed analysis (30+ parameters)

    • Overcome spectral overlap limitations of conventional flow cytometry

• Advanced microscopy applications:

  • Super-resolution microscopy:

    • Apply STORM or PALM techniques to visualize REG4 distribution below diffraction limit

    • Achieve 20-30 nm spatial resolution of REG4 localization

    • Map REG4 interaction with membrane microdomains

  • Intravital microscopy:

    • Track REG4-expressing cells in living organisms

    • Monitor dynamic changes in REG4 expression during disease progression

    • Assess drug delivery and target engagement in real-time

• Microfluidic and organ-on-chip systems:

  • Integration methodology:

    • Incorporate FITC-conjugated REG4 antibody staining in microfluidic channels

    • Perform on-chip immunofluorescence with reduced reagent consumption

    • Enable continuous monitoring of REG4 expression in 3D culture systems

  • Applications:

    • Study REG4 regulation in physiologically relevant microenvironments

    • Assess heterogeneity of REG4 expression in tumor models

    • Screen therapeutic candidates with improved predictability

• Computational biology integration:

  • Machine learning for image analysis:

    • Train neural networks to identify REG4-positive cell populations

    • Automate quantification across large dataset collections

    • Extract complex phenotypic features beyond intensity measurements

  • Systems biology approaches:

    • Integrate REG4 protein data with multi-omics datasets

    • Model REG4 regulatory networks and signaling pathways

    • Predict therapeutic targets through in silico analysis

These technological combinations are expanding our understanding of REG4 biology and opening new avenues for diagnostic and therapeutic applications .

How can FITC-conjugated REG4 antibodies be optimized for analysis of clinical specimens?

Optimizing FITC-conjugated REG4 antibodies for clinical specimen analysis requires specific methodological considerations:

• Formalin-fixed, paraffin-embedded (FFPE) tissue processing:

  • Antigen retrieval optimization:

    • Test multiple buffers: citrate (pH 6.0), EDTA (pH 8.0), and Tris-EDTA (pH 9.0)

    • Compare heat-induced (95-100°C, 20 minutes) vs. enzymatic methods

    • Assess microwave, pressure cooker, and water bath heating approaches

  • Protocol modifications:

    • Extend permeabilization time (0.3% Triton X-100, 30 minutes)

    • Increase primary antibody incubation (overnight at 4°C)

    • Use tyramide signal amplification for low-abundance detection

    • Include Sudan Black B treatment (0.1% in 70% ethanol) to reduce autofluorescence

• Frozen tissue section optimization:

  • Preservation of antigenicity:

    • Fix briefly with 1-2% paraformaldehyde (10 minutes)

    • Block extensively with 10% normal serum + 1% BSA (1 hour)

    • Apply antibody at optimized concentration (typically 1-5 μg/mL)

  • Technical considerations:

    • Maintain consistent section thickness (6-8 μm optimal)

    • Allow complete drying before fixation

    • Include detergent (0.1% Triton X-100) in antibody diluent

• Circulating tumor cell and liquid biopsy applications:

  • Cell enrichment strategies:

    • Optimize density gradient or immunomagnetic separation

    • Standardize fixation timing post-collection (within 2 hours)

  • Staining protocol:

    • Implement multimarker approach (REG4-FITC + epithelial markers + CD45 exclusion)

    • Minimize washing steps to reduce cell loss

    • Use nuclear counterstain for cell identification

• Standardization for clinical correlation:

  • Quantification approach:

    • Develop scoring system (e.g., H-score: intensity × percentage positive cells)

    • Establish threshold values for positivity based on control populations

    • Use digital image analysis for objective quantification

  • Quality control measures:

    • Include positive and negative control tissues on each slide

    • Incorporate internal control cells with known REG4 expression

    • Implement dual-observer scoring for validation

These optimizations ensure reliable and reproducible analysis of REG4 expression in clinical specimens, which is essential for biomarker development and patient stratification studies .