REG4 Antibody, Biotin conjugated

What is REG4 and why is it a significant research target?

REG4 is a 17-18 kDa secreted glycoprotein belonging to the calcium-dependent lectin superfamily and regenerating gene family. It is physiologically expressed in the gastrointestinal tract, particularly in normal colon mucosa, and becomes significantly upregulated in several pathological conditions including colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel diseases (Crohn's disease and ulcerative colitis) . Outside the GI tract, REG4 upregulation has been observed in prostate adenocarcinoma . From a cellular perspective, REG4 marks a distinct population of deep secretory cells at the bottom of colon crypts, where it's believed to serve as an analogous support system for LGR5+ stem cells, similar to how Paneth cells support stem cells in the upper GI tract . While its precise physiological function remains under investigation, REG4 appears to play roles in inflammatory and metaplastic responses of the GI epithelium .

What are the key differences between standard REG4 antibodies and biotin-conjugated versions?

Biotin-conjugated REG4 antibodies offer several distinct advantages over unconjugated versions. The primary benefit is their enhanced detection sensitivity through the strong biotin-avidin interaction system, which provides significant signal amplification. Standard REG4 antibodies require a secondary antibody for detection, while biotinylated versions can directly bind to streptavidin-coupled detection systems . This eliminates one step in immunoassays and reduces background from species cross-reactivity. Biotin conjugation allows for greater flexibility in detection methods, as the same biotinylated primary antibody can be used with various streptavidin-conjugated reporters (HRP, fluorophores, gold particles) . Additionally, biotinylated antibodies are particularly valuable in multiplex immunoassays where simultaneous detection of multiple targets is required, as they allow for clean separation of detection signals without cross-reactivity between secondary antibodies .

What is the recommended dilution range for REG4 biotinylated antibodies in different applications?

The optimal dilution of REG4 biotinylated antibodies varies by application type and detection system. For Western blotting applications, REG4 antibodies typically work effectively at dilutions ranging from 1:500 to 1:2000 . For immunohistochemistry (IHC), the recommended dilution range is typically 1:50 to 1:500 . When working with biotin-conjugated antibodies specifically, the working dilution may need optimization, but typical protocols recommend diluting concentrated biotin conjugate antibody (100×) at 1:100 with biotin-conjugate antibody diluent before use . For ELISA applications, antibody concentrations must be carefully titrated to achieve optimal signal-to-noise ratios within the detection range (typically 1.57-100 ng/mL for REG4) . It's strongly recommended to perform preliminary experiments with a dilution series to determine the optimal concentration for each specific experimental system, as antibody performance can vary based on sample type, detection method, and target abundance .

How should I design validation experiments to confirm the specificity of a REG4 biotinylated antibody?

Designing rigorous validation experiments for REG4 biotinylated antibodies requires multiple complementary approaches. First, conduct positive and negative control experiments using cells/tissues known to express REG4 (such as colon cancer cells or normal colon mucosa) versus those that don't . For a comprehensive validation, perform antibody testing across multiple applications: Western blotting using cell lines with known REG4 expression (like SGC-7901 cells) , immunohistochemistry on human colon cancer tissue , and ELISA with recombinant REG4 protein .

To confirm specificity, use a blocking peptide competition assay where pre-incubation of the antibody with its specific immunogen should abolish signal. Additionally, employ genetic validation by testing the antibody on REG4 knockout/knockdown samples compared to wild-type controls. For biotinylated antibodies specifically, include controls to assess endogenous biotin interference by running parallel assays with detection systems in the absence of the primary antibody . Finally, cross-reactivity testing is crucial - while the antibody may be designed for human REG4, testing its reactivity with mouse REG4 (67% sequence homology) can help establish species specificity . Document all validation data systematically, including positive and negative controls, and specific experimental conditions.

How can I optimize dual staining protocols using REG4 biotinylated antibodies alongside other markers?

Optimizing dual staining protocols with REG4 biotinylated antibodies requires careful planning to prevent signal interference and cross-reactivity. Begin by establishing single-staining protocols for each antibody individually before attempting co-staining. For REG4 biotinylated antibody paired with another primary antibody, select detection systems that are compatible and distinguishable - for example, using streptavidin-HRP with a chromogenic substrate for REG4 detection, and a fluorescently labeled secondary antibody for the other marker .

To minimize background and non-specific binding, include thorough blocking steps not only for endogenous peroxidase activity but also for endogenous biotin, particularly in biotin-rich tissues like liver, kidney, and brain. Use avidin/biotin blocking kits followed by protein blocking solutions containing serum from the same species as secondary antibodies . Sequential staining rather than simultaneous incubation often yields cleaner results - complete the staining for one marker before beginning the second. If studying REG4-positive secretory cells in colon crypts alongside stem cell markers such as LGR5, optimize antigen retrieval conditions that work for both epitopes (e.g., TE buffer pH 9.0 works well for REG4) . Finally, include appropriate controls: single-stained sections, secondary-only controls, and isotype controls to distinguish true co-localization from artifactual staining or bleed-through.

What strategies should I employ to troubleshoot weak or inconsistent signals when using REG4 biotinylated antibodies?

When encountering weak or inconsistent signals with REG4 biotinylated antibodies, implement a systematic troubleshooting approach. First, evaluate antibody integrity by confirming proper storage conditions (typically -20°C with glycerol) and checking for signs of degradation . Next, optimize antibody concentration - weak signals may require higher concentrations within the recommended range (1:50-1:500 for IHC, 1:500-1:2000 for WB) .

For Western blotting applications, ensure adequate protein loading (20-30 μg total protein), and verify transfer efficiency. Since REG4 is a relatively small protein (14-18 kDa), adjust gel percentage and transfer conditions accordingly . For IHC applications, antigen retrieval is critical - test both citrate buffer (pH 6.0) and TE buffer (pH 9.0), as the latter is often recommended for REG4 . When using biotinylated antibodies specifically, ensure thorough blocking of endogenous biotin using avidin/biotin blocking kits prior to primary antibody incubation.

Check reagent quality and freshness, particularly detection substrates which can deteriorate over time. If working with tissue samples, optimize fixation time - overfixation can mask epitopes. For ELISA applications, ensure sample pH is neutral, as extreme pH can affect antibody-antigen binding . Additionally, extend antibody incubation times (overnight at 4°C rather than 1-2 hours) or implement signal amplification systems like tyramide signal amplification to enhance weak signals. Finally, consider tissue-specific expression levels - REG4 is heterogeneously expressed and may require optimization for specific sample types .

What is the optimal sample preparation method for detecting REG4 in different gastrointestinal tissue types?

Optimal sample preparation for REG4 detection varies between gastrointestinal tissue types due to differential expression patterns and tissue characteristics. For all GI tissues, fixation in 10% neutral buffered formalin for 24-48 hours provides good epitope preservation, though prolonged fixation should be avoided as it can mask REG4 epitopes . For colon tissues, where REG4 marks deep secretory cells at the bottom of crypts, orientation during embedding is crucial to ensure proper visualization of the crypt architecture .

For paraffin sections, 4-5 μm thickness is ideal, and antigen retrieval using TE buffer at pH 9.0 is recommended for optimal REG4 detection, though citrate buffer (pH 6.0) can also be effective . For frozen sections, brief fixation in cold acetone improves antibody binding while preserving tissue morphology. When preparing protein lysates for Western blotting, RIPA buffer supplemented with protease inhibitors effectively extracts REG4 while preserving its epitopes .

For challenging samples like pancreatic tissue, which contains high levels of endogenous enzymes, rapid fixation and processing are essential to prevent autolysis. When preparing samples for ELISA, tissue homogenization should be performed in PBS with protease inhibitors, followed by clarification through centrifugation . Additionally, for secreted REG4 in cell culture supernatants, concentration via ultrafiltration may be necessary to achieve detectable levels within the assay's range (1.57-100 ng/mL) . Regardless of sample type, inclusion of positive control tissues (known to express REG4) such as colon cancer tissue or SGC-7901 cells is essential for validating preparation protocols .

How can I quantitatively assess REG4 expression levels using biotinylated antibodies in tissue microarrays?

Quantitative assessment of REG4 expression in tissue microarrays (TMAs) using biotinylated antibodies requires a standardized approach combining appropriate staining methods with rigorous image analysis. Begin with consistent IHC protocol optimization using the recommended dilution range (1:50-1:500) and antigen retrieval with TE buffer pH 9.0 . Include positive controls (colon cancer tissue) and negative controls (primary antibody omission) on each TMA slide.

For quantification, digital image analysis provides the most objective assessment. Scan stained TMAs using a digital slide scanner and apply specialized image analysis software to quantify staining intensity and percentage of positive cells. Implement a scoring system such as H-score (combining intensity [0-3] with percentage of positive cells [0-100%]) or Allred score for consistent evaluation . For biotinylated antibodies specifically, it's crucial to thoroughly block endogenous biotin using avidin/biotin blocking kits to prevent false-positive results, particularly in biotin-rich tissues .

To ensure reproducibility, have at least two independent observers score a subset of cores, calculating inter-observer agreement (kappa statistic). Additionally, perform calibration using quantitative standards - either recombinant REG4 protein at known concentrations or reference tissue samples with established expression levels. For correlation with clinical outcomes, categorize expression levels (negative, low, moderate, high) based on established cutoffs in the literature for REG4 in gastrointestinal or inflammatory conditions . Finally, validate TMA findings by performing whole-section analysis on a subset of cases to account for tumor heterogeneity.

What are the recommended storage conditions and stability parameters for maintaining REG4 biotinylated antibody activity?

Maintaining optimal activity of REG4 biotinylated antibodies requires careful attention to storage conditions and handling procedures. For long-term storage, keep antibodies at -20°C in manufacturer-supplied buffer, typically containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Under these conditions, antibodies remain stable for approximately one year from the date of shipment . Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt into single-use volumes - this is particularly important for biotinylated antibodies as biotin conjugation can be sensitive to repeated temperature fluctuations.

For working solutions, store at 4°C and use within 1-2 weeks. The presence of sodium azide as a preservative in storage buffers helps prevent microbial contamination but should be noted as it can inhibit HRP activity in some detection systems . For diluted biotin-conjugated antibody working solutions, prepare fresh on the day of use rather than storing diluted antibody . When handling the antibody, minimize exposure to light, particularly for fluorescently labeled streptavidin conjugates used for detection.

Stability can be monitored through regular performance checks against positive control samples. A gradual reduction in signal intensity or increased background may indicate antibody deterioration. Additionally, some manufacturers recommend performing a quick centrifugation of the product prior to opening to gather liquid at the bottom of the vial . For biotinylated antibodies specifically, avoid buffers containing free biotin which can compete with the antibody for streptavidin binding. Finally, maintain records of antibody lot numbers, receipt dates, and performance characteristics to track potential variations between batches .

How do expression patterns of REG4 differ across gastrointestinal cancers, and what methodological approaches best capture these differences?

REG4 expression patterns show distinct profiles across various gastrointestinal cancers, requiring tailored methodological approaches for accurate characterization. In colorectal cancer, REG4 is significantly upregulated compared to normal mucosa, particularly in mucinous and poorly differentiated subtypes . For detecting this overexpression, IHC using optimized protocols (1:50-1:500 dilution with TE buffer pH 9.0 for antigen retrieval) provides spatial information about REG4 distribution within the tumor microenvironment .

In gastric adenocarcinoma, REG4 expression correlates with certain histological subtypes, especially those with intestinal metaplasia. Western blotting (1:500-1:2000 dilution) of tissue lysates can quantitatively compare REG4 levels between tumor subtypes . For pancreatic cancer, where REG4 may serve as a serum biomarker, ELISA methods with a detection range of 1.57-100 ng/mL offer advantages for quantifying circulating protein levels .

To accurately capture these tissue-specific differences, multi-modal approaches are recommended. Combining tissue-based methods (IHC and in situ hybridization for localization) with quantitative techniques (Western blotting and qRT-PCR for expression levels) provides comprehensive characterization . For comparing expression across different GI cancers within the same study, tissue microarrays with standardized staining protocols offer controlled comparison. Additionally, laser capture microdissection before protein or RNA extraction ensures cell type-specific analysis, particularly important given REG4's expression in specific secretory cell populations . Finally, correlating REG4 expression with established molecular subtypes of each cancer type (MSI status in colorectal cancer, HER2 status in gastric cancer) provides context for interpreting expression differences .

What are the technical considerations when investigating REG4's role in inflammatory bowel disease using biotinylated antibodies?

Investigating REG4's role in inflammatory bowel disease (IBD) using biotinylated antibodies presents several technical challenges requiring specific methodological considerations. First, tissue sampling strategy is crucial - paired biopsies from inflamed and non-inflamed regions provide internal controls, while sampling from different disease segments (ileum versus colon) helps map expression patterns across the intestinal tract . When processing IBD tissues, rapid fixation is essential to preserve REG4 epitopes while preventing degradation from high protease activity in inflamed samples.

For IHC applications in IBD tissues, background reduction is particularly important due to high infiltration of biotin-rich immune cells. Implement stringent blocking protocols using avidin/biotin blocking kits followed by protein blocking with serum before applying REG4 biotinylated antibodies . The recommended dilution range of 1:50-1:500 for IHC should be carefully titrated for IBD samples, often requiring lower dilutions (1:50-1:100) for optimal signal-to-noise ratio .

To distinguish REG4 expression in epithelial versus immune cells, dual immunofluorescence staining combining REG4 detection with lineage-specific markers (E-cadherin for epithelium, CD45 for immune cells) is recommended. When examining REG4's potential as a biomarker in IBD, both tissue and serum analysis should be performed - ELISA methods with sensitivity below 0.78 ng/mL are suitable for detecting subtle changes in REG4 levels . Additionally, compare REG4 expression with established IBD biomarkers like calprotectin and CRP for contextual interpretation.

For longitudinal studies monitoring REG4 during disease flares and remission, standardized collection protocols are essential to minimize technical variation. Finally, correlate REG4 expression patterns with specific IBD phenotypes (Crohn's versus ulcerative colitis) and disease activity scores to elucidate its potential role in disease pathogenesis or as a biomarker of inflammation .

How can I design experiments to investigate the functional relationship between REG4 and LGR5+ stem cells in colonic crypts?

Designing experiments to investigate the functional relationship between REG4 and LGR5+ stem cells in colonic crypts requires multi-faceted approaches spanning spatial, functional, and molecular analyses. Begin with high-resolution spatial characterization using dual immunofluorescence staining with biotinylated REG4 antibody (1:50-1:100 dilution) and LGR5 antibody to map their relative positions within colonic crypts . Confocal microscopy with z-stack imaging will reveal the three-dimensional relationship between REG4+ secretory cells and LGR5+ stem cells.

For functional studies, organoid culture systems provide powerful tools. Establish colonic organoids from mice expressing fluorescent reporters for LGR5 and REG4, then use FACS to isolate pure populations of each cell type. Co-culture experiments comparing LGR5+ stem cell growth with or without REG4+ cells will reveal supportive functions. Additionally, supplement organoid media with recombinant REG4 protein at various concentrations to assess direct effects on stem cell maintenance and differentiation .

For mechanistic insights, perform secretome analysis of REG4+ cells using mass spectrometry to identify secreted factors beyond REG4 itself. Conduct transcriptome analysis (RNA-seq) comparing LGR5+ stem cells cultured with or without REG4+ cells/recombinant REG4 to identify key responsive pathways. Employ CRISPR-Cas9 to knock out REG4 in colonic epithelial cells, then assess effects on stem cell niche formation and function .

In vivo validation is essential - utilize lineage tracing in mouse models with inducible labeling of REG4+ cells to track potential conversion to stem cells during injury repair. Finally, examine pathological conditions - compare the REG4-LGR5 relationship in normal colon versus inflammatory bowel disease and colorectal cancer tissues using optimized IHC protocols with proper antigen retrieval in TE buffer pH 9.0 . This comprehensive approach will elucidate whether REG4+ deep secretory cells truly function analogously to Paneth cells in supporting the stem cell niche of colonic crypts .

What are the strengths and limitations of using REG4 biotinylated antibodies in multiplex detection systems?

Multiplex detection systems incorporating REG4 biotinylated antibodies offer several advantages but also present technical challenges that require careful consideration. The primary strength is the versatility of biotin-streptavidin interactions, which enable signal amplification through multiple streptavidin-coupled detection systems without species cross-reactivity issues inherent to secondary antibody approaches . This facilitates cleaner discrimination between multiple markers in complex tissue environments such as the intestinal epithelium.

Another advantage is detection flexibility - the same REG4 biotinylated antibody can be visualized using various streptavidin conjugates (fluorophores, enzymes, quantum dots) depending on the experimental requirements . For studying REG4's relationship with other proteins in gastrointestinal pathology, this adaptability is particularly valuable. Additionally, the high sensitivity of biotin-streptavidin systems (10-15 fold higher than direct detection) helps visualize REG4 in samples with low expression levels .

When designing multiplex panels including REG4, consider compatible antigen retrieval conditions - while REG4 detection works optimally with TE buffer pH 9.0, other markers may require different conditions . Finally, spectral overlap in fluorescent multiplex systems can complicate analysis, requiring careful selection of fluorophores and potentially necessitating spectral unmixing during image analysis.

How can I validate the recovery and linearity of a REG4 ELISA assay using biotinylated detection antibodies?

Validating recovery and linearity of REG4 ELISA assays using biotinylated detection antibodies requires systematic experimental approaches to ensure reliable quantification across diverse sample types. For recovery validation, prepare spike-recovery experiments by adding known concentrations of recombinant REG4 protein to various relevant matrices (cell culture media, serum, tissue homogenates). The spiked samples should span the assay's detection range (1.57-100 ng/mL) . Calculate recovery percentages by comparing measured concentrations to expected values - acceptable recovery typically falls between 80-120% across sample types .

For linearity assessment, prepare serial dilutions (typically 1:2, 1:4, 1:8, and 1:16) of samples containing high REG4 concentrations. Plot the measured concentration against the dilution factor, and calculate the percentage of observed to expected concentration at each dilution. Acceptable linearity is indicated by percentages consistently between 80-120% across the dilution series . To ensure robustness, perform these validations across multiple sample types relevant to REG4 research (serum, cell culture media, colon tissue homogenates) .

Additional technical considerations include optimizing working dilutions of biotinylated antibodies - dilute concentrated biotin conjugate antibody (100×) 1:100 with appropriate diluent before use . When calculating results, use computer software capable of generating four-parameter logistic (4-PL) curve-fit analysis for the standard curve, as this best accommodates the sigmoidal relationship in immunoassays .

For comprehensive validation, also assess precision through intra-plate (samples tested multiple times on one plate) and inter-plate (samples tested across multiple plates) variability, targeting coefficient of variation (CV) values <10% for intra-assay and <15% for inter-assay variability . Finally, determine assay sensitivity by calculating the minimum detectable dose (MDD) - adding two standard deviations to the mean optical density of zero standard replicates and determining the corresponding concentration (typically <0.78 ng/mL for REG4) . This systematic validation ensures reliable quantification of REG4 across diverse experimental contexts.

What considerations are important when developing automated image analysis algorithms for quantifying REG4 expression in tissue sections?

Developing robust automated image analysis algorithms for REG4 quantification in tissue sections requires addressing several technical and biological considerations. First, standardize image acquisition parameters including magnification (typically 20× or 40×), exposure settings, and white balance calibration to ensure consistent input for the algorithm . For optimal performance with REG4 biotinylated antibodies, capture images of sections stained using the recommended IHC protocol (1:50-1:500 dilution, TE buffer pH 9.0 antigen retrieval) .

Algorithm development should begin with tissue segmentation to distinguish epithelial regions (where REG4 is primarily expressed) from stromal and inflammatory components. Incorporate morphological features specific to the tissue type being analyzed - for colon tissue, identify crypt structures where REG4-positive deep secretory cells reside . Implement color deconvolution to separate the REG4 signal (typically DAB chromogen) from counterstain (hematoxylin) and potential background staining.

For cellular-level analysis, include nuclear segmentation as a first step, followed by cytoplasmic/membrane identification to properly localize REG4, which is primarily cytoplasmic and secreted . Design the algorithm to quantify multiple parameters: percentage of positive cells, staining intensity (weak/moderate/strong), and histological distribution patterns (diffuse versus focal) . For multiplex images containing REG4 alongside other markers, implement channel separation and co-localization analysis.

Validation is crucial - compare algorithm results against manual scoring by pathologists on a training set of diverse tissue samples, then test performance on an independent validation set. Calculate correlation coefficients and evaluate concordance using Cohen's kappa statistic. Ensure the algorithm performs consistently across different tissue types where REG4 is studied (colon, pancreas, gastric tissue) .

Finally, implement quality control measures - the algorithm should flag images with staining artifacts, insufficient tissue, or excessive background for manual review. Provide confidence scores with automated quantification results and design the system to learn from expert corrections through machine learning approaches. This comprehensive development approach will yield reliable automated quantification of REG4 expression patterns in diverse research and potentially clinical applications.

What are the emerging research applications for REG4 biotinylated antibodies beyond current established uses?

Emerging research applications for REG4 biotinylated antibodies extend beyond traditional detection methods into innovative diagnostic and therapeutic domains. One promising frontier is liquid biopsy development - REG4 biotinylated antibodies can enable ultrasensitive detection of circulating REG4 in blood samples using technologies like single-molecule arrays (Simoa) or digital ELISA, potentially providing minimally invasive biomarkers for gastrointestinal cancers and inflammatory conditions . The high sensitivity of biotin-streptavidin systems makes them particularly suitable for detecting low-abundance REG4 in circulation.

In spatial biology, REG4 biotinylated antibodies are being integrated into highly multiplexed imaging platforms like imaging mass cytometry and multiplexed ion beam imaging (MIBI), allowing simultaneous visualization of REG4 alongside dozens of other markers to map cellular neighborhoods within the intestinal epithelium . This helps define the relationship between REG4+ secretory cells and other niche components in unprecedented detail.

For therapeutic applications, REG4 biotinylated antibodies are being explored for targeted drug delivery systems, where antibody-drug conjugates selectively deliver cytotoxic payloads to REG4-overexpressing tumor cells in colorectal and pancreatic cancers . Additionally, biotinylated antibodies facilitate immuno-PET imaging when conjugated with radiolabeled streptavidin, enabling non-invasive visualization of REG4 expression in patients.

In organoid research, REG4 biotinylated antibodies are being employed for live-cell sorting of specific cellular populations to establish enriched organoid cultures representing distinct epithelial lineages . Finally, in the emerging field of engineered cellular therapies, REG4-targeting approaches may help direct immunotherapeutic cells to gastrointestinal tumors that overexpress this protein . These diverse applications highlight how biotinylated REG4 antibodies are expanding beyond conventional research tools to enable transformative approaches in gastrointestinal cancer diagnosis and treatment.

How might future technological developments enhance or modify current REG4 detection methodologies?

Future technological developments will likely transform REG4 detection methodologies across multiple dimensions. Single-cell proteomics approaches will revolutionize our understanding of REG4 expression heterogeneity - mass cytometry (CyTOF) coupled with REG4 biotinylated antibodies will enable quantification at single-cell resolution alongside dozens of other markers, revealing previously undetectable subpopulations within REG4-expressing cells . Nanoscale imaging technologies like super-resolution microscopy (STORM, PALM) will visualize the subcellular localization and trafficking of REG4 with unprecedented precision, potentially revealing new aspects of its secretory pathways.

Advances in microfluidic technologies will enable continuous monitoring of REG4 secretion from live cells or organoids in real-time, providing dynamic information about its regulation under various stimuli or drug treatments . For clinical applications, automated digital pathology platforms incorporating artificial intelligence will standardize REG4 quantification in tissue samples, reducing inter-observer variability and enabling more precise correlation with patient outcomes .

Emerging biosensor technologies utilizing aptamer-based recognition elements coupled with REG4 antibodies will enable continuous monitoring of REG4 levels in biological fluids or cell culture systems with improved sensitivity and temporal resolution compared to conventional ELISA . Additionally, CRISPR-based reporters that produce luminescent or fluorescent signals in response to REG4 binding will facilitate live-cell imaging of REG4-receptor interactions.

In tissue analysis, spatial transcriptomics combined with protein detection (e.g., Digital Spatial Profiling) will correlate REG4 protein expression with comprehensive transcriptional profiles in the same tissue section, providing unprecedented insights into the molecular microenvironment of REG4-expressing cells . Finally, the development of recombinant nanobodies against REG4 may provide smaller detection reagents with improved tissue penetration for imaging applications and potentially enable intravital microscopy of REG4 dynamics in animal models. These technological advances collectively promise to transform our ability to detect, quantify, and functionally characterize REG4 in both research and clinical contexts.

What are the most significant unresolved questions regarding REG4's function that could be addressed using biotinylated antibody-based approaches?

Several critical unresolved questions regarding REG4's function could be addressed through strategic application of biotinylated antibody-based approaches. First, the precise molecular mechanism by which REG4+ deep secretory cells support LGR5+ stem cells in the colon remains unclear . Using biotinylated REG4 antibodies in proximity ligation assays could identify direct protein-protein interactions between REG4 and potential receptors on stem cells, while pull-down experiments with biotinylated antibodies followed by mass spectrometry could characterize the complete REG4 interactome in intestinal epithelial cells.

The signaling pathways activated by REG4 remain poorly defined. Combining REG4 detection with phospho-specific antibodies in multiplexed immunofluorescence or CyTOF could map the activation of downstream signaling molecules in response to REG4 stimulation . Additionally, the role of REG4 in the tumor microenvironment beyond cancer cells themselves remains unexplored - multiparameter immunohistochemistry with biotinylated REG4 antibodies alongside immune cell markers could reveal potential immunomodulatory functions.

How REG4 expression is dynamically regulated during intestinal inflammation, injury, and repair represents another critical question . Time-course studies in models of colitis or radiation injury, using biotinylated REG4 antibodies for tissue analysis, could map expression patterns relative to key phases of the injury-repair cycle. Furthermore, the potential role of REG4 as a secreted biomarker for early detection or monitoring of gastrointestinal malignancies could be investigated using highly sensitive digital ELISA platforms with biotinylated antibodies for detection .