RCN3 Antibody

What is RCN3 and why is it important for research?

RCN3 (Reticulocalbin 3) is a calcium-binding protein of approximately 37.5 kDa that resides primarily in the endoplasmic reticulum (ER) lumen. It belongs to the CREC protein family and functions as a molecular chaperone assisting in protein biosynthesis and transport within the ER .

RCN3 has emerged as an important research target due to its:

• Involvement in various pathological conditions including emphysema/COPD

• Potential role as a biomarker in multiple cancer types

• Contribution to pulmonary surfactant homeostasis

• Regulatory function in protein biosynthesis through the secretory pathway

Studies have shown RCN3 is widely expressed across numerous tissue types, making it relevant for research across multiple physiological systems .

How should I select the appropriate RCN3 antibody for my specific experimental application?

Selection should be guided by your experimental requirements:

For critical research, validate reactivity against your specific species. While human and mouse reactivity is commonly validated, predicted reactivity for rat and bovine samples should be experimentally confirmed .

What are the optimal storage and handling conditions for RCN3 antibodies?

RCN3 antibodies require specific storage and handling protocols to maintain functionality:

• Short-term storage (up to 2 weeks): Maintain at 2-8°C in refrigeration

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Buffer composition: Typically supplied in PBS with 0.09% (w/v) sodium azide

• Shipping conditions: Usually shipped on wet ice

Research demonstrates that repeated freeze-thaw cycles significantly reduce antibody binding efficiency. Data from stability studies suggest limiting to ≤5 freeze-thaw cycles to preserve specificity and sensitivity in experimental applications .

What protocols yield optimal results for RCN3 detection in immunohistochemistry?

Based on published research methods, the following protocol has demonstrated reliable results:

• Tissue preparation:

  • Fix tissues in 10% phosphate-buffered formalin for ≥48 hours

  • Embed in paraffin and section at 4 μm thickness

• Antigen retrieval:

  • Apply sodium citrate method under high pressure for 5 minutes

  • Incubate sections in 3% hydrogen peroxide for 30 minutes

• Antibody application:

  • Block sections for 30 minutes

  • Incubate with primary anti-RCN3 antibody (1:20-1:50 dilution) overnight at 4°C

  • Apply secondary antibody for 30 minutes

  • Visualize with DAB reagent

  • Counterstain nuclei with hematoxylin

For quantitative analysis, studies have utilized mean optical density calculations using quantitative dynamic program analysis systems such as Image Pro Plus, with three random views selected per IHC image to calculate optical density of positive cells divided by total cell area .

How can I optimize Western blot protocols for RCN3 detection?

RCN3 Western blot optimization requires attention to several critical parameters:

• Sample preparation:

  • For cellular samples: Use RIPA buffer supplemented with protease inhibitors

  • For tissue homogenates: Homogenize in ice-cold buffer containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, and 50 mM Tris-HCl (pH 7.4)

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation around the 37.5 kDa range

  • Load 20-40 μg of total protein per lane for cell lysates

• Transfer and detection:

  • Transfer to PVDF membranes at 100V for 90 minutes (wet transfer)

  • Block with 5% non-fat milk in TBST for 1 hour

  • Incubate with anti-RCN3 antibody (1:2000 dilution) overnight at 4°C

  • Use HRP-conjugated secondary antibodies (1:5000 dilution)

  • Develop using enhanced chemiluminescence reagents

Research data shows that reducing agent concentration can affect RCN3 band specificity due to its EF-hand calcium-binding domains. Optimization may be required for different sample types .

How can I validate RCN3 antibody specificity to ensure experimental reliability?

Multi-level validation approaches are recommended:

• Positive and negative tissue controls:

  • Use lung tissue as positive control (high endogenous expression)

  • Include knockout/knockdown samples as negative controls

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide (134-163 amino acids from central region for some antibodies)

  • Compare signal between blocked and unblocked antibody

• Cross-reactivity assessment:

  • Test against recombinant RCN3 protein

  • Evaluate potential cross-reactivity with other CREC family members

  • Compare multiple antibodies targeting different epitopes

• Orthogonal method verification:

  • Correlate protein expression with mRNA levels using qRT-PCR

  • Verify subcellular localization matches known ER distribution pattern

Research demonstrates that RCN3 antibody validation through CRISPR/Cas9 knockout models provides definitive specificity confirmation in cancer research applications .

How can RCN3 antibodies be utilized to investigate its role in cancer progression?

Recent research has established methodological approaches for studying RCN3 in cancer contexts:

• Expression correlation studies:

  • Use RCN3 antibodies for IHC analysis of tumor microarrays

  • Correlate expression with clinical parameters (stage, grade, survival)

  • Apply semi-quantitative scoring systems for comparative analysis

• Functional investigations:

  • Combine RCN3 knockdown approaches (shRNA or CRISPR/Cas9) with antibody-based detection

  • Evaluate changes in proliferation, migration, and invasion

  • Measure alterations in immune-related factors including TGFβ1, IL-10, and IL-6

• Mechanistic pathway analysis:

  • Implement co-immunoprecipitation with RCN3 antibodies to identify protein interaction partners

  • Perform immunofluorescence co-localization studies with ER markers

  • Assess impact on ribosomal and translation pathways

Research in glioblastoma has demonstrated that RCN3 knockdown significantly enhances survival in orthotopic mouse models, suggesting therapeutic potential. RNA-seq analysis revealed that RCN3 knockdown altered expression of genes related to translation, ribosome function, stem cell differentiation, and extracellular matrix .

What methodological approaches can be used to study RCN3's role in pulmonary diseases?

Research into RCN3's role in pulmonary pathologies employs several antibody-dependent techniques:

• Quantitative tissue analysis:

  • Implement immunohistochemistry on lung sections from COPD/emphysema patients and controls

  • Measure mean optical density of RCN3-positive cells

  • Correlate with morphometric measurements (mean linear intercept) of alveolar damage

• Animal model validation:

  • Analyze RCN3 expression in cigarette smoke-exposed and elastase-induced emphysema models

  • Compare with selective Rcn3 knockout in alveolar epithelial type II cells (AECIIs)

  • Examine impact on pulmonary surfactant-associated proteins (SP-A, SP-D) and lipid transporter ABCA3

• Molecular interaction studies:

  • Investigate ER stress pathways using antibodies against RCN3 and GRP78

  • Assess collagen secretion and matrix metalloproteinase expression

  • Evaluate apoptotic markers including cleaved caspase-3

Data from human COPD patients showed significantly higher RCN3 expression (p<0.05) compared to controls, with similar findings in mouse models. Selective deletion of Rcn3 in AECIIs resulted in significant remission of emphysematous changes in response to elastase, demonstrating its potential role in disease progression .

How can I design multiplex immunofluorescence experiments to study RCN3 in relation to ER stress and the secretory pathway?

Multiplex immunofluorescence requires careful experimental design:

• Antibody selection and validation:

  • Choose primary antibodies from different host species (e.g., rabbit anti-RCN3 with mouse anti-GRP78)

  • Validate each antibody individually before multiplexing

  • Test for cross-reactivity between secondary antibodies

• Sample preparation optimization:

  • Use 4% paraformaldehyde fixation for 15-20 minutes

  • Optimize permeabilization (0.1-0.3% Triton X-100 for 5-15 minutes)

  • Include antigen retrieval if necessary for formalin-fixed tissues

• Staining protocol:

  • Apply sequential staining for multiple primary antibodies

  • Use tyramide signal amplification for detection of low-abundance proteins

  • Include appropriate controls (single stains, no primary controls, fluorophore minus controls)

• Analysis methods:

  • Implement confocal microscopy with spectral unmixing

  • Quantify colocalization using Pearson's correlation coefficient

  • Apply spatial analysis to map protein distribution within the ER

Research demonstrates that RCN3 colocalizes significantly with ER stress markers during pathological conditions, with Pearson's correlation coefficients of 0.65-0.78 reported in pulmonary disease models .

What strategies can address non-specific binding or weak signal issues with RCN3 antibodies?

Systematic troubleshooting approaches for common issues:

• Non-specific binding in Western blots:

  • Increase blocking time (2-3 hours with 5% BSA instead of milk)

  • Titrate primary antibody concentrations (test 1:1000 to 1:5000 dilutions)

  • Increase washing stringency (0.1-0.3% Tween-20 in TBS)

  • Use gradient gels (8-16%) to better resolve proteins around 37.5 kDa

• Weak signal in immunohistochemistry:

  • Optimize antigen retrieval (test citrate pH 6.0 vs. EDTA pH 9.0 buffers)

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

  • Implement signal amplification systems (ABC or polymer-based detection)

  • Test fresh tissue vs. archived samples for epitope preservation

• Background in immunofluorescence:

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

  • Pre-absorb secondary antibodies with tissue powder

  • Prepare antibody dilutions fresh before use

  • Consider photobleaching autofluorescence (especially in tissues with elastin/collagen)

Research indicates that certain RCN3 epitopes may be masked by posttranslational modifications including N-glycosylation, potentially requiring deglycosylation treatment for consistent detection in certain applications .

How can I verify if contradictory RCN3 expression data is due to antibody limitations or biological variation?

When facing contradictory results, implement this verification strategy:

• Antibody comparison:

  • Test multiple antibodies targeting different RCN3 epitopes

  • Compare polyclonal vs. monoclonal antibody performance

  • Evaluate lot-to-lot variation using reference samples

• Multi-omics validation:

  • Correlate protein levels with mRNA expression (qRT-PCR)

  • Verify with orthogonal techniques (Western blot, IHC, and flow cytometry)

  • Consider absolute quantification using recombinant protein standards

• Biological context assessment:

  • Evaluate RCN3 expression across different stages of disease progression

  • Consider tissue heterogeneity and microenvironmental factors

  • Assess impact of experimental conditions (hypoxia, ER stress, calcium flux)

• Technical controls:

  • Include positive control samples with known RCN3 expression

  • Implement spike-in controls to assess recovery efficiency

  • Perform parallel analysis of housekeeping proteins

Research demonstrates that RCN3 expression varies significantly across different cancer stages, with up to 3.42-fold increases observed in certain disease models compared to controls (p<0.001) .

What quality control metrics should be established for RCN3 antibody-based assays in a research laboratory?

Implement these quality control measures for robust research data:

• Antibody characterization:

  • Document lot number, source, and target epitope information

  • Establish working dilution ranges for each application

  • Determine antibody stability timeline at recommended storage conditions

• Assay performance metrics:

  • Calculate signal-to-noise ratios for each application

  • Establish reproducibility parameters (intra-assay and inter-assay CV%)

  • Determine limits of detection and quantification in quantitative applications

• Reference standards:

  • Maintain positive control lysates/tissues with verified RCN3 expression

  • Include calibration curves using recombinant RCN3 protein

  • Implement internal controls for normalization

• Validation documentation:

  • Record antibody validation experiments (Western blot, IHC, knockdown verification)

  • Document experimental protocols including critical parameters

  • Maintain antibody performance tracking over time