REG1B Antibody

What is REG1B and why is it a significant research target?

REG1B (Regenerating Islet-Derived 1 Beta) is a member of the regenerating gene family involved in pancreatic regeneration and inflammatory processes. This protein, also known as lithostathine-1-beta, pancreatic stone protein 2, and several other aliases, has gained research significance due to its role in tissue regeneration and potential as a biomarker for various pathological conditions . REG1B is expressed in the pancreas and gastrointestinal tissues, with emerging evidence suggesting its involvement in inflammatory diseases, pancreatic disorders, and certain gastrointestinal cancers. The study of REG1B through reliable antibody-based detection methods provides insights into its expression patterns, functional mechanisms, and potential clinical applications.

What are the common applications for REG1B antibodies in research?

REG1B antibodies are primarily employed in several key research applications:

• Western Blotting: Used to detect and quantify REG1B protein expression in cell or tissue lysates. The recommended dilution for Western blotting ranges from 0.01-3 μg/mL, depending on the specific antibody and sample characteristics .

• Immunohistochemistry (IHC): Enables visualization of REG1B protein localization in tissue sections. IHC applications typically require antibody concentrations of 5-30 μg/mL .

• Direct ELISA: Allows quantitative measurement of REG1B in biological samples. Some REG1B antibodies show cross-reactivity with related proteins, which should be considered during experimental design .

• Immunocytochemistry (ICC): Enables subcellular localization studies of REG1B in cultured cells .

Each application requires specific optimization of antibody dilution, incubation conditions, and detection methods to achieve optimal results while minimizing background and non-specific binding.

How should REG1B antibodies be stored and handled to maintain reactivity?

Proper storage and handling of REG1B antibodies is crucial for maintaining their specificity and sensitivity over time:

• Storage Temperature: Store at 4°C for frequent use (short-term) or aliquot and store at -20°C for long-term storage (up to 24 months) .

• Freeze-Thaw Cycles: Avoid repeated freeze/thaw cycles as these can significantly reduce antibody activity and specificity. Preparing single-use aliquots is recommended for antibodies stored at -20°C .

• Reconstitution: For lyophilized antibodies, reconstitute at the recommended concentration (e.g., 0.2 mg/mL in sterile PBS for some products) .

• Stability: Under proper storage conditions, the loss rate should be less than 5% within the expiration date. Stability can be verified through accelerated thermal degradation testing (e.g., incubation at 37°C for 48h) .

• Working Solutions: When preparing working dilutions, use sterile techniques and appropriate buffer systems as recommended in the product documentation.

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifetime of these research reagents.

What controls should be included when using REG1B antibodies in Western blotting?

Robust experimental design for Western blotting with REG1B antibodies requires several essential controls:

• Positive Control: Include a known positive sample, such as recombinant human REG1B protein (Gln23-Asn166) expressed in E. coli . This helps validate antibody detection capability and serves as a reference for expected signal size.

• Negative Control: Use samples known not to express REG1B or those from REG1B-knockout models. Alternatively, include secondary antibody-only controls to assess non-specific binding.

• Loading Control: Implement housekeeping protein detection (e.g., β-actin, GAPDH) to normalize REG1B expression levels and ensure equal sample loading.

• Molecular Weight Marker: Include a reliable protein ladder to confirm the expected molecular weight of detected REG1B (approximately 16-18 kDa).

• Antibody Specificity Control: When feasible, perform pre-absorption of the antibody with the target antigen to confirm signal specificity.

Including these controls helps distinguish specific from non-specific signals and provides a framework for reliable data interpretation and reproducibility across experimental replicates.

How can I optimize immunohistochemistry protocols for REG1B detection in different tissue types?

Optimizing IHC protocols for REG1B detection requires systematic adjustment of multiple parameters:

• Tissue Type Considerations:

  • Human stomach tissue shows strong REG1B expression and serves as an excellent positive control

  • Pancreatic tissue exhibits specific REG1B localization patterns

  • Cancer tissues may display altered expression requiring protocol modifications

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended for formalin-fixed tissues

  • Enzymatic retrieval may be appropriate for certain tissue types

  • Optimization is needed for tissues with high endogenous biotin or peroxidase activity

• Antibody Concentration:

  • Start with 5-30 μg/mL concentration range

  • Perform titration experiments to determine optimal signal-to-noise ratio

  • Consider longer incubation times at lower concentrations for reduced background

• Detection Systems:

  • HRP-linked secondary antibodies (e.g., HRP-Linked Caprine Anti-Rabbit IgG) at approximately 2 μg/mL concentration work well for REG1B visualization

  • DAB (3,3'-diaminobenzidine) substrate provides good visualization of REG1B in stomach and pancreatic tissues

  • Consider fluorescent detection for co-localization studies

• Counterstaining:

  • Hematoxylin provides good nuclear contrast for DAB-based detection

  • DAPI works well for fluorescent detection systems

Systematic optimization of these parameters will yield reproducible and specific REG1B staining across different tissue types.

What are the recommended blocking agents for minimizing background in REG1B antibody applications?

Selection of appropriate blocking agents is crucial for reducing non-specific binding and background signals:

• Western Blotting:

  • 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) is effective for most applications

  • 3-5% BSA (bovine serum albumin) may yield cleaner results with phospho-specific antibodies

  • Consider specialized blocking buffers for tissues with high endogenous biotin

• Immunohistochemistry:

  • Normal serum (5-10%) from the species in which the secondary antibody was raised

  • Commercial blocking solutions containing both proteins and detergents

  • For fluorescent applications, consider auto-fluorescence quenchers

• ELISA:

  • 1-3% BSA in PBS is generally effective

  • Specialized blocking buffers may be required to minimize cross-reactivity with related proteins

• Specific Considerations for REG1B:

  • Due to documented cross-reactivity with mouse Reg1 (approximately 20%) and rat Reg1 (approximately 5%) , additional blocking steps may be necessary when working with these species

  • Pre-absorption with unrelated Reg family proteins may help increase specificity

Optimizing blocking conditions specifically for each application will significantly improve signal-to-noise ratios and data reliability.

How can I assess REG1B antibody cross-reactivity with other REG family proteins?

Cross-reactivity assessment is essential when working with REG1B antibodies due to the high sequence homology among REG family members:

• Quantitative Cross-Reactivity Analysis:

  • Direct ELISA using purified recombinant proteins can quantify cross-reactivity

  • For goat anti-human REG1B antibodies, approximately 20% cross-reactivity with recombinant mouse Reg1, 5% with mouse Reg2 and rat Reg1, and less than 1% with human Reg4 and mouse Reg3A has been documented

  • Competitive binding assays can determine relative affinity for different REG proteins

• Western Blot Cross-Reactivity Assessment:

  • Compare band patterns using recombinant REG family proteins

  • Analyze samples from knockout models lacking specific REG proteins

  • Pre-absorption with related proteins can confirm antibody specificity

• Cross-Reactivity Mitigation Strategies:

  • Epitope mapping to identify unique REG1B regions

  • Affinity purification against specific REG1B epitopes

  • Competitive blocking with related REG proteins

Understanding the cross-reactivity profile is critical for accurate data interpretation, particularly in multi-species studies or when examining tissues with expression of multiple REG family members.

What approaches can be used to study REG1B expression in disease models?

Investigation of REG1B in pathological conditions requires tailored methodological approaches:

• Differential Expression Analysis:

  • Comparing REG1B expression between normal and diseased tissues (e.g., stomach cancer vs. normal stomach)

  • Quantitative Western blotting with normalization to housekeeping proteins

  • IHC scoring methods to assess changes in expression patterns and localization

• Temporal Expression Studies:

  • Time-course experiments during disease progression

  • Correlation with clinical parameters and disease severity

  • Inducible expression systems to model dysregulated REG1B expression

• Functional Studies:

  • siRNA/shRNA knockdown to assess functional consequences of REG1B reduction

  • Overexpression models to investigate effects of elevated REG1B

  • Co-immunoprecipitation to identify disease-specific interaction partners

• Multiplexed Analysis:

  • Combination of REG1B antibodies with markers of inflammation, cell proliferation, or other disease-relevant proteins

  • Sequential immunostaining for co-localization studies

  • Integration with other methodologies (e.g., in situ hybridization) for mRNA-protein correlation

These approaches provide comprehensive insights into the role of REG1B in disease pathophysiology and potential therapeutic targeting.

How can REG1B antibodies be utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence enables simultaneous visualization of REG1B alongside other markers:

• Antibody Selection Considerations:

  • Choose REG1B antibodies raised in different host species than other target antibodies

  • If using multiple rabbit-derived antibodies, consider directly conjugated formats or sequential immunostaining

  • Verify spectral compatibility of selected fluorophores to avoid bleed-through

• Staining Protocol Development:

  • Optimize single-color staining protocols individually before multiplexing

  • Determine optimal antibody concentrations to achieve comparable signal intensities

  • Consider tyramide signal amplification for low-abundance targets

• Available Conjugated Formats:

  • REG1B antibodies are available conjugated to various fluorophores including Alexa Fluor 488, 555, 594, 647, and 750

  • These pre-conjugated formats eliminate the need for species-specific secondary antibodies

  • Direct conjugation reduces background from cross-reactivity between secondary antibodies

• Controls for Multiplex Studies:

  • Single-color controls to establish detection thresholds and compensation settings

  • Fluorescence-minus-one (FMO) controls to set gating boundaries

  • Absorption controls to verify absence of spectral overlap

Multiplex approaches provide spatial context for REG1B expression relative to cell types, signaling molecules, and tissue structures, yielding richer biological insights than single-marker studies.

What strategies can address weak or absent REG1B signals in Western blotting?

When encountering weak or absent REG1B signals, systematic troubleshooting can identify and resolve the underlying issues:

• Sample Preparation Optimization:

  • Ensure complete protein extraction using appropriate lysis buffers

  • Add protease inhibitors to prevent REG1B degradation during sample processing

  • Consider enrichment methods for low-abundance samples

• Antibody-Related Adjustments:

  • Increase antibody concentration (starting recommendation: 0.1 μg/mL)

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

  • Verify antibody activity using positive control samples (recombinant REG1B)

• Detection System Enhancement:

  • Implement more sensitive detection systems (chemiluminescent vs. colorimetric)

  • Use signal amplification methods for low-abundance targets

  • Optimize exposure times for digital imaging systems

• Transfer Efficiency Improvement:

  • Adjust transfer conditions for small proteins (~16-18 kDa)

  • Consider semi-dry transfer systems for enhanced transfer of small proteins

  • Verify transfer efficiency using reversible protein stains

• Antibody Selection Considerations:

  • Different REG1B antibodies target distinct epitopes (N-terminal vs. full-length)

  • Consider switching to an antibody validated specifically for Western blotting

  • Evaluate alternative host species options (rabbit vs. goat polyclonal antibodies)

Systematic implementation of these strategies typically resolves detection issues for most research applications.

How can I validate the specificity of REG1B antibody staining in immunohistochemistry?

Validation of antibody specificity is essential for reliable IHC data interpretation:

• Positive and Negative Tissue Controls:

  • Human stomach and pancreas tissues serve as positive controls

  • Use tissues known not to express REG1B as negative controls

  • Include tissues from REG1B knockout models when available

• Peptide Competition Assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare staining patterns between blocked and unblocked antibody

  • Specific staining should be significantly reduced or eliminated

• Multiple Antibody Validation:

  • Compare staining patterns using antibodies targeting different REG1B epitopes

  • Consistent localization patterns across antibodies suggest specific detection

  • N-terminal antibodies and full-length antibodies should show similar patterns

• Correlation with Alternative Detection Methods:

  • Compare IHC results with in situ hybridization for REG1B mRNA

  • Correlate with Western blot analysis of the same tissues

  • Verify with mass spectrometry-based protein identification when possible

• Technical Controls:

  • Include secondary antibody-only controls

  • Implement isotype controls matched to the primary antibody

  • Process serial sections with and without primary antibody

What are the common causes of cross-reactivity when using REG1B antibodies?

Understanding sources of cross-reactivity is important for accurate data interpretation:

• Structural Homology with REG Family Members:

  • REG1B shares significant sequence homology with other REG proteins

  • Documented cross-reactivity includes approximately 20% with mouse Reg1 and 5% with mouse Reg2 and rat Reg1

  • Selection of antibodies targeting unique regions can minimize this issue

• Species-Specific Considerations:

  • Human REG1B antibodies may show variable cross-reactivity with mouse and rat homologs

  • Cross-reactivity profiles should be verified experimentally for each application

  • Host species may influence cross-reactivity patterns (rabbit vs. goat polyclonal antibodies)

• Antibody Production Methods:

  • Polyclonal antibodies (available from rabbit and goat hosts) recognize multiple epitopes

  • Immunization protocols influence epitope recognition patterns

  • Affinity purification methods affect antibody specificity profiles

• Experimental Conditions Affecting Specificity:

  • Fixation methods can alter epitope accessibility and cross-reactivity profiles

  • Buffer conditions and blocking agents influence non-specific binding

  • Antibody concentration inversely correlates with specificity

Understanding these factors allows researchers to implement appropriate controls and interpret results with appropriate caution regarding potential cross-reactive signals.

How are REG1B antibodies being used in biomarker development research?

REG1B antibodies are increasingly utilized in biomarker research across multiple disease contexts:

• Diagnostic Biomarker Applications:

  • Detection of altered REG1B expression in pancreatic and gastrointestinal disorders

  • Development of immunoassays for REG1B quantification in biological fluids

  • Correlation of REG1B levels with disease severity and progression

• Tissue-Based Biomarker Research:

  • IHC evaluation of REG1B expression patterns in cancer tissues

  • Investigation of REG1B as a prognostic marker in gastrointestinal malignancies

  • Correlation with pathological features and clinical outcomes

• Technological Approaches:

  • Multiplex protein arrays incorporating REG1B antibodies

  • Development of point-of-care testing platforms

  • Integration with other biomarkers for improved diagnostic accuracy

• Validation Requirements:

  • Analytical validation of antibody performance in clinical sample types

  • Establishment of reference ranges and cutoff values

  • Correlation with gold standard diagnostic methods

These applications highlight the translational potential of REG1B antibodies beyond basic research contexts and underscore the importance of thorough antibody validation.

What considerations are important when developing REG1B antibody-based assays?

Development of robust REG1B assays requires attention to several critical parameters:

• Antibody Pair Selection for Sandwich Assays:

  • Choose antibodies recognizing non-overlapping epitopes

  • Evaluate detection limits and dynamic range with recombinant standards

  • Assess performance in the specific sample matrix of interest

• Cross-Reactivity Management:

  • Characterize cross-reactivity with related REG family proteins

  • Implement blocking strategies to minimize non-specific binding

  • Consider absorption steps to remove cross-reactive antibodies

• Sample Processing Considerations:

  • Optimize sample collection and storage conditions

  • Evaluate need for sample pre-treatment (heat inactivation, delipidation)

  • Determine appropriate dilution factors for different sample types

• Assay Validation Parameters:

  • Establish precision (intra- and inter-assay)

  • Determine accuracy through spike-recovery experiments

  • Evaluate analytical sensitivity and specificity

• Reference Material Development:

  • Utilize recombinant REG1B with confirmed sequence and structure

  • Consider development of stable reference standards

  • Implement quality control procedures for long-term monitoring

Careful attention to these factors supports development of reliable REG1B immunoassays suitable for research and potential clinical applications.

What novel techniques are emerging for REG1B detection and functional analysis?

Recent methodological advances are expanding the toolkit for REG1B research:

• Proximity Ligation Assays (PLA):

  • Detection of REG1B protein-protein interactions in situ

  • Visualization of specific post-translational modifications

  • Single-molecule sensitivity for low-abundance interactions

• Mass Cytometry Applications:

  • Integration of REG1B detection in CyTOF panels

  • Single-cell analysis of REG1B expression in heterogeneous populations

  • Correlation with signaling pathway activation markers

• CRISPR-Based Functional Studies:

  • Genome editing to create REG1B knockout and knockin models

  • CRISPRa/CRISPRi for modulation of REG1B expression

  • Base editing for introduction of specific mutations

• Structural Biology Approaches:

  • Antibody epitope mapping using hydrogen-deuterium exchange mass spectrometry

  • Cryo-EM studies of REG1B complexes

  • Structure-guided antibody development targeting functional domains

• Spatial Transcriptomics Integration:

  • Correlation of REG1B protein detection with spatial gene expression

  • Multi-omic approaches for comprehensive functional characterization

  • Tissue ecosystem analysis of REG1B-expressing cells

These emerging approaches provide unprecedented resolution for understanding REG1B biology and pathological alterations, establishing foundations for future therapeutic targeting.