GUCA2B Antibody

What is GUCA2B and what is its significance in intestinal research?

GUCA2B, also known as Guanylate Cyclase Activator 2B (Uroguanylin), is a peptide involved in intestinal fluid secretion regulation. This protein functions as an endogenous ligand for guanylate cyclase-C (GC-C or GUCY2C), activating intracellular signaling pathways that influence intestinal homeostasis.

Research significance includes:

• Regulation of intestinal fluid secretion

• Potential involvement in irritable bowel syndrome

• Role in colorectal cancer investigations

• Part of co-regulatory networks with other intestinal proteins (AQP8, SPIB)

• Expression in specialized intestinal epithelial cell populations

The protein has gained research interest as studies have revealed its expression in specific cell populations and its potential involvement in multiple intestinal pathologies .

Which cell types express GUCA2B in the intestine?

The cellular localization of GUCA2B has been a subject of ongoing investigation with several key findings:

• In human and rat duodenum and human colon, GUCA2B/Guca2b is expressed in dispersed solitary epithelial cells, some with tuft cell-like morphology

• GUCA2B is not co-expressed with CHGA in human duodenal cells, suggesting enteroendocrine (EC) cells are not the major source

• In mouse models, Guca2b expression is enriched in cells that also express Guca2a (guanylin)

• Recent single-cell studies have characterized GUCA2B-expressing cells in the terminal ileum and adjacent colon

Notably, epithelial cells displaying intense Guca2b expression stand out distinctly from the rest of the epithelial cell population . The exact identity of these cells remains under investigation, with evidence suggesting they may represent specialized secretory cells distinct from classical enteroendocrine cells.

What applications are GUCA2B antibodies validated for?

GUCA2B antibodies have been validated for multiple research applications:

Different antibodies may demonstrate variable performance across applications. For example, some GUCA2B antibodies work optimally for Western blotting while others are better suited for immunohistochemistry . Researchers should review validation data specific to their application before selecting an antibody.

How should I select the appropriate GUCA2B antibody for my research?

When selecting a GUCA2B antibody, consider these critical factors:

• Species reactivity: Ensure the antibody recognizes GUCA2B from your species of interest (human, mouse, rat)

• Clonality: Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide greater specificity

• Application validation: Verify the antibody has been validated for your specific application (WB, IHC, ELISA)

• Immunogen information: Antibodies raised against different regions of GUCA2B may have different specificities (e.g., AA 22-106, AA 37-112)

• Host species: Consider potential cross-reactivity issues in your experimental system

• Conjugation: Determine if you need an unconjugated antibody or one conjugated with a detection tag (HRP, FITC, biotin)

• Published validation: Prioritize antibodies with validation in peer-reviewed literature

For example, Proteintech Group's 18113-1-AP (polyclonal) has been referenced in published studies for multiple applications including Western blot, ELISA, ICC, and IHC .

What are the technical considerations for Western blotting with GUCA2B antibodies?

Successful Western blotting for GUCA2B requires careful attention to these technical parameters:

• Sample preparation: Use EDTA-free protease inhibitor cocktails during protein extraction to preserve GUCA2B integrity

• Buffer selection: Some GUCA2B antibodies perform better with PBST (PBS with 0.1% Tween 20), while others require TBST (TBS with 0.1% Tween 20)

• Antibody dilutions: Primary antibody dilutions typically range from 1:500 to 1:1000; anti-GUCA2B should be diluted 500-fold with TBST in some protocols

• Secondary antibody: Use goat anti-rabbit IgG-conjugated HRP at dilutions of 1:2000 to 1:10000

• Loading controls: GAPDH serves as a reliable internal control for normalization

• Detection systems: ECL substrates with chemiluminescent detection systems provide optimal visualization

Optimization may be required for specific experimental conditions. For instance, in co-regulatory network studies of GUCA2B with SPIB and AQP8, researchers used a 1:500 dilution of anti-GUCA2B with TBST and 1:10000 dilution of secondary antibody .

How can I optimize immunohistochemistry protocols for GUCA2B detection?

For optimal GUCA2B immunohistochemical detection:

• Tissue fixation: Appropriate fixation is critical for preserving epitope structure while maintaining tissue morphology

• Antigen retrieval: Optimize based on specific antibody recommendations, as some epitopes may require heat-induced or enzymatic retrieval

• Blocking: Use appropriate blocking solutions (typically 5-10% normal serum) to minimize background

• Antibody selection: Choose antibodies specifically validated for IHC applications

• Incubation conditions: Typically overnight at 4°C for primary antibodies

• Visualization method: Select fluorescent or chromogenic detection based on research needs

It's noteworthy that some researchers have encountered difficulties with GUCA2B immunohistochemical detection. For example, one study reported being "unable to detect specific immunohistochemical staining with the monoclonal antibodies isolated during the development of the new immunoassay in human intestinal tissue slices" . This highlights the importance of antibody validation and protocol optimization.

What challenges exist in detecting GUCA2B at the cellular level?

Several challenges in detecting GUCA2B expression are evident from the literature:

• Variable expression patterns: GUCA2B shows focal or dispersed expression, making detection challenging

• Low abundance: Detection may require highly sensitive methods in certain tissues

• Specificity issues: Some antibodies may show non-specific binding

• Technical limitations: Multiple studies report difficulties with immunohistochemical detection

• Species differences: Variations in GUCA2B sequence and expression between species affect antibody performance

Alternative approaches have been developed to address these challenges:

• In situ hybridization (ISH) for GUCA2B mRNA detection provides greater sensitivity

• Transgenic reporter models (e.g., using Venus fluorescent protein under Guca2a promoter control) enable live cell identification

• Single-cell RNA sequencing approaches help characterize GUCA2B-expressing cell populations

How does GUCA2B expression vary across different intestinal regions?

Research demonstrates significant regional differences in GUCA2B expression:

• In rat colon, Guca2b was described as "focally expressed"

• In human and rat duodenum and human colon, GUCA2B/Guca2b appears in "dispersed solitary epithelial cells"

• RNA-seq analysis showed transcriptomic differences in GUCA2B-expressing cells between different intestinal regions

• Principal component analysis demonstrated that GUCA2B-expressing cells cluster by tissue of origin, indicating regional specialization

• Duodenal GUCA2B-expressing cells are transcriptomically distinct from their counterparts in jejunum and ileum

• Strong similarities exist between GUCA2B-expressing cells from the ileum and jejunum compared to those from the duodenum

These regional differences should guide experimental design when studying GUCA2B expression patterns, as antibody performance may vary across different intestinal segments.

What controls should be included when using GUCA2B antibodies?

Rigorous controls are essential for GUCA2B antibody experiments:

Positive controls:

• Tissues with known GUCA2B expression (intestinal tissues)

• Recombinant GUCA2B protein for Western blot validation

Negative controls:

• Secondary antibody only (omitting primary antibody)

• Isotype control antibodies

• Tissues or cells known not to express GUCA2B

• Competing peptide controls to demonstrate specificity

Validation controls:

• siRNA or CRISPR knockout of GUCA2B to demonstrate specificity

• Multiple antibodies targeting different epitopes of GUCA2B

• Correlation of protein detection with mRNA expression data (e.g., comparing with in situ hybridization)

Internal controls:

• Housekeeping proteins (e.g., GAPDH for Western blotting)

• Known cell type markers for comparative localization in tissue sections

How can GUCA2B antibodies be used in co-localization studies?

For effective co-localization studies involving GUCA2B:

• Select compatible antibodies:

  • Choose GUCA2B antibodies and other marker antibodies raised in different host species

  • Consider directly conjugated primary antibodies to avoid cross-reactivity

• Design multi-label protocols:

  • Sequential staining protocols may be necessary

  • Optimize antibody dilutions for each marker

  • Select fluorophores with minimal spectral overlap

• Include appropriate markers:
  Research has investigated co-localization of GUCA2B with:

  • Enteroendocrine cell markers like CHGA

  • Tuft cell markers like Dclk1

  • Goblet cell markers (Muc2, Tff3, Spink4)

  • Enterocyte markers (Adh6a, Khk, Alpi, Slc16a3, Batf2)

  • GUCY2C (GC-C receptor) which has been found to be co-expressed with GUCA2B in some tissues

• Overcome technical limitations:

  • When antibody compatibility is limited, use serial sections with adjoining sides facing up on slides

  • Consider in situ hybridization for GUCA2B mRNA detection combined with immunohistochemistry for protein markers

How can I study the co-regulatory network of GUCA2B with other proteins?

To investigate GUCA2B's co-regulatory networks:

• Take a multi-omics approach:

  • Combine transcriptomics, proteomics, and functional studies

  • Analyze correlated expression patterns in single-cell RNA-seq data

  • Validate protein-level interactions using co-immunoprecipitation

• Focus on known interacting partners:
  Research has identified a co-regulatory network involving:

  • AQP8 (Aquaporin 8)

  • SPIB (transcription factor)

  • GUCY2C (the receptor for GUCA2B)

  • GUCA2A (Guanylin, another ligand for GUCY2C)

• Implement experimental validation:

  • Use siRNA or CRISPR to knockdown GUCA2B and assess effects on partner genes

  • Employ Western blotting to measure protein levels of GUCA2B, AQP8, and SPIB simultaneously

  • Utilize dual immunolabeling to confirm co-expression at the cellular level

• Combine in vitro and in silico methods:

  • As demonstrated in the literature, investigating co-regulatory networks of AQP8, GUCA2B, and SPIB in colorectal cancer benefits from both computational and experimental approaches

What are the advantages of transgenic models in GUCA2B research?

Transgenic models offer several advantages over antibody-based detection methods:

• Enhanced visualization:

  • Fluorescent protein reporters enable direct visualization without antibody staining

  • The literature describes a transgenic mouse model expressing Venus fluorescent protein under Guca2a promoter control

  • This approach allowed FACS sorting of fluorescent cells for detailed analysis

• Validation capabilities:

  • RT-qPCR confirmed enrichment of both Guca2a and Guca2b in fluorescent cell populations isolated from transgenic mice

  • This approach validated the co-expression of these related genes in specific cell populations

• Multi-omics integration:

  • Transgenic models enable isolation of live cells for transcriptomic analysis

  • RNA-seq on sorted populations revealed 515 genes enriched in Guca2a-positive cells (which also express Guca2b) across four intestinal regions

  • Principal component analysis showed clustering by both tissue of origin and reporter expression

• Overcoming antibody limitations:

  • Particularly valuable when researchers are "unable to detect specific immunohistochemical staining with monoclonal antibodies"

  • Allows investigation of GUCA2B-expressing cells when antibody-based methods prove challenging

What dilution ranges are optimal for GUCA2B antibodies?

Optimal dilution ranges for GUCA2B antibodies vary by application:

Western Blotting:

• Primary antibody: 1:500 to 1:1000 dilution

• Secondary antibody: 1:2000 to 1:10000 dilution

Immunohistochemistry:

• Specific recommendations vary by antibody

• Some manufacturers indicate dilution information in product documentation

ELISA:

• Coating antibody concentrations typically range from 1-10 μg/ml

• Detection antibody dilutions are often more dilute than for Western blotting

As stated in commercial documentation, "Optimal dilutions/concentrations should be determined by the end user" . Performing titration experiments is essential to determine the optimal concentration for your specific sample type and detection system. Different lots of the same antibody may require re-optimization.

How can I validate the specificity of a GUCA2B antibody?

Comprehensive validation of GUCA2B antibody specificity should include:

• Control tissues and cells:

  • Test tissues with known GUCA2B expression patterns (intestinal tissues)

  • Include tissues known not to express GUCA2B as negative controls

• Molecular validation:

  • Knockdown or knockout GUCA2B expression and confirm signal reduction

  • Peptide competition assays to block specific binding

• Complementary detection methods:

  • Compare protein detection with mRNA localization using in situ hybridization

  • Use multiple antibodies targeting different epitopes of GUCA2B

  • Correlate with reporter systems (e.g., transgenic models)

• Characteristic verification:

  • Confirm expected molecular weight in Western blots

  • Verify cellular localization consistent with known biology

  • Compare expression patterns with published literature

• Cross-reactivity assessment:

  • Test against related proteins (e.g., GUCA2A)

  • Verify species specificity if working across multiple species

What are the differences between human and mouse GUCA2B antibodies?

Key differences between human and mouse GUCA2B antibodies include:

• Immunogen specificity:

  • Human GUCA2B antibodies: Often raised against synthetic human Uroguanylin

  • Mouse GUCA2B antibodies: Generated against recombinant mouse GUCA2B protein (amino acids 22-106)

• Cross-reactivity limitations:

  • Species-specific antibodies typically show limited cross-reactivity

  • Antibody catalogs clearly specify reactivity as either human or mouse

  • Few antibodies demonstrate validated cross-species reactivity

• Application profiles:

  • Human GUCA2B antibodies: Commonly employed in clinical research and human cell lines

  • Mouse GUCA2B antibodies: Primarily used with mouse models and experimental systems

• Epitope recognition:

  • Different epitopes may be targeted in human versus mouse antibodies

  • Antibodies target specific amino acid ranges (e.g., AA 37-112, AA 22-106)

  • These differences could affect detection of processed forms or splice variants