REG4 Antibody, HRP conjugated

What is REG4 and what cellular functions does it regulate?

REG4 (also known as Regenerating islet-derived protein 4, GISP, or RELP) is a secreted protein primarily expressed in the gastrointestinal tract and normal colon mucosa. It plays significant roles in several biological processes including:

• Supporting LGR5+ stem cell populations in the colon crypts

• Promoting cell proliferation and resistance to apoptosis

• Involvement in inflammatory responses in bowel diseases

• Contributing to chemoresistance mechanisms in various cancers

REG4 marks a population of deep secretory cells at the bottom of crypts in the colon. While Paneth cells support LGR5+ stem cells in the upper gastrointestinal tract by providing signals like Wnt3, EGF, and Notch ligands, the colon lacks these cells. Research suggests that REG4+ cells serve as an analogous support system for the LGR5+ stem cell population in the colon .

What applications is REG4 Antibody, HRP conjugated suitable for?

REG4 Antibody, HRP conjugated is specifically designed for the following applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): Primary application with optimized sensitivity for REG4 detection in biological samples

• Serological analysis: Can be used in sandwich ELISA formats for detecting REG4 in human serum and plasma samples

• Immunoblotting: Though Western blot is not the primary application for the HRP-conjugated version, basic immunoblotting can be performed with appropriate protocol modifications

The antibody's HRP conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and potentially reducing background noise in detection systems .

What is the molecular weight of REG4 detected by antibodies?

When using REG4 antibodies for research applications such as Western blotting, researchers should expect to detect a protein band at approximately 14 kDa, which corresponds to the molecular weight of human REG4 . This information is crucial for properly identifying REG4 in experimental results and distinguishing it from non-specific binding or cross-reactive proteins.

What are the recommended protocols for sample preparation when using REG4 Antibody, HRP conjugated?

For optimal REG4 detection using HRP-conjugated antibodies, the following sample preparation protocols are recommended:

For tissue sections (IHC):

• Use immersion-fixed paraffin-embedded sections (particularly effective for colon cancer tissue)

• Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-basic prior to antibody incubation

• Incubate with REG4 polyclonal antibody at a concentration of 10 μg/mL overnight at 4°C

• Develop using HRP-DAB detection system and counterstain with hematoxylin for optimal visualization

For ELISA applications:

• Use freshly collected samples when possible

• For serum/plasma detection, dilute samples appropriately in assay buffer (typically 1:2 to 1:10 depending on expected concentration)

• Include purified recombinant REG4 protein as standards for quantification

• Block non-specific binding sites thoroughly to minimize background signals

Following these protocols ensures reliable and reproducible detection of REG4 in various sample types.

What dilutions and buffer systems are recommended for REG4 Antibody, HRP conjugated?

The optimal working conditions for REG4 Antibody, HRP conjugated vary by application:

For ELISA applications:

• Recommended dilution: 1:1000 to 1:5000 in appropriate assay buffer

• Buffer composition: PBS (pH 7.4) with 0.05% Tween-20 and 1-5% BSA or casein as a blocking agent

For tissue staining:

• Dilution range: 1:200-1:500 for immunohistochemistry applications

• Buffer system: TBS or PBS with 0.05% Tween-20 and appropriate blocking components

The antibody is provided in a storage buffer containing 50% Glycerol, 0.01M PBS (pH 7.4), with 0.03% Proclin 300 as a preservative . This formulation maintains stability during freeze-thaw cycles and provides optimal activity in the recommended applications.

How should REG4 Antibody, HRP conjugated be stored to maintain activity?

To preserve the functionality and specificity of REG4 Antibody, HRP conjugated:

• Upon receipt, store immediately at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles which can diminish antibody activity and lead to increased background signal

• For working solutions, store at 4°C for up to one week

• Protect from light exposure, as HRP conjugates are sensitive to photobleaching

• Do not dilute the stock antibody until ready for use to prevent degradation

Following these storage recommendations will ensure optimal antibody performance throughout your experimental timeline.

How can REG4 Antibody be used to investigate the mechanisms of chemoresistance in colorectal cancer?

REG4 antibodies provide valuable tools for exploring chemoresistance mechanisms in colorectal cancer through several methodological approaches:

• Expression correlation studies:

  • Compare REG4 protein levels in chemoresistant versus chemosensitive tumor samples using immunohistochemistry

  • Research has revealed that REG4 protein is strongly expressed in chemoresistant rectal cancer cells but weakly expressed in drug-sensitive cells

• Pathway analysis:

  • Investigate REG4's role in activating the EGFR-PI3K-Akt-NF-κB pathway using co-immunoprecipitation and phosphorylation studies

  • REG4 has been shown to promote proliferation, antiapoptosis, migration, and invasion of DLD-1 cells by activating this pathway

• Lipid metabolism studies:

  • Examine how REG4 affects lipid droplet assembly (but not de novo lipogenesis) in relation to chemoresistance

  • Research indicates REG4 inhibits transcription of acetyl-CoA carboxylase 1 (ACC1) and ATP-citrate lyase (ACLY) by disassociating complex formation in their promoters

• Stimulation experiments:

  • Analyze how REG4 expression is stimulated by factors like TNFα, EGF, basic fibroblast growth factor, and hepatocyte growth factor

  • These stimulatory factors can promote cell proliferation and resistance to H₂O₂-induced apoptosis

These approaches using REG4 antibodies can help elucidate the multifaceted role of REG4 in chemoresistance and potentially identify novel therapeutic targets.

What is the significance of the inverse correlation between REG4 mRNA and protein expression in colorectal cancer?

One of the intriguing aspects of REG4 biology is the discrepancy between mRNA and protein expression patterns in colorectal cancer:

These findings highlight the complexity of REG4 biology and the importance of multimodal analysis when studying its role in cancer progression.

How can REG4 antibodies be applied to investigate stem cell dynamics in gastrointestinal tissues?

REG4 antibodies can serve as valuable tools for exploring stem cell populations in gastrointestinal tissues through several experimental approaches:

• Lineage tracing studies:

  • Use REG4 immunostaining to identify and track deep secretory cells at the bottom of colon crypts

  • Combine with LGR5+ stem cell markers to investigate the support network for intestinal stem cells

  • Research has demonstrated that REG4+ cells serve an analogous support role to Paneth cells in the upper GI tract

• Organoid culture applications:

  • Apply REG4 antibodies for immunostaining to identify unique populations of secretory progenitor cells in intestinal stem cell-derived organoid cultures

  • This approach has been validated in human induced pluripotent stem cells and intestinal organoids

• Signaling pathway analysis:

  • Investigate the relationship between REG4+ cells and canonical stem cell signaling pathways (Wnt, Notch, EGF)

  • Explore how REG4 expression influences stem cell maintenance and differentiation in normal and pathological conditions

• Differential expression studies:

  • Compare REG4 expression patterns across the progression from normal tissue to metaplasia, adenoma, and cancer

  • Research shows REG4 expression gradually decreases from gastric intestinal metaplasia and adenoma to cancer and gastritis

These applications highlight the utility of REG4 antibodies in understanding the complex dynamics of stem cell populations in gastrointestinal tissues.

What factors might affect REG4 detection specificity in tissue samples?

When working with REG4 antibodies for tissue analysis, several factors can influence detection specificity:

• Epitope accessibility issues:

  • Heat-induced epitope retrieval is critical for proper REG4 detection in FFPE tissues

  • Insufficient antigen retrieval can lead to false-negative results

  • Optimal REG4 detection requires using Antigen Retrieval Reagent-basic and incubation at 10 μg/mL overnight at 4°C

• Cross-reactivity considerations:

  • Ensure the antibody has been validated for human REG4 specificity (particularly important as REG4 belongs to a family of related proteins)

  • Use properly validated antibodies with >95% purity, such as Protein G purified preparations

• Expression heterogeneity:

  • REG4 expression can vary significantly across different regions of the same tumor

  • Consider analyzing multiple tissue sections or using tissue microarrays to account for this heterogeneity

  • REG4 staining is typically localized to the cytoplasm of epithelial cells in colon tissues

• Technical variables:

  • Fixation duration and conditions can significantly impact epitope preservation

  • Section thickness (4-5 μm recommended) affects staining intensity and specificity

  • Incubation times and temperatures must be carefully controlled

  • Background reduction requires proper blocking and washing steps

Addressing these factors ensures reliable and reproducible REG4 detection in tissue samples.

How can researchers validate REG4 antibody specificity for their specific experimental systems?

Rigorous validation of REG4 antibody specificity is essential for reliable experimental results:

• Positive and negative controls:

  • Use human colon cancer tissue as a positive control for REG4 expression, where specific labeling should be localized to the cytoplasm of epithelial cells

  • Include tissues known to lack REG4 expression as negative controls

  • Consider using cell lines with confirmed REG4 expression (e.g., DLD-1 cells) versus those with low/no expression

• Peptide competition assays:

  • Pre-incubate the REG4 antibody with purified recombinant human REG4 protein before application

  • Specific binding should be significantly reduced or eliminated

  • Recombinant human REG4 protein spanning Asp23-Pro158 (Accession # Q9BYZ8) can be used for this purpose

• Multiple antibody approach:

  • Confirm results using alternative REG4 antibodies targeting different epitopes

  • Compare staining patterns between polyclonal and monoclonal antibodies

  • For example, compare rabbit polyclonal antibodies with goat anti-human REG4 antibodies

• Correlation with other detection methods:

  • Validate immunohistochemistry findings with Western blot analysis

  • Consider mRNA expression analysis (noting the inverse correlation in certain contexts)

  • Complement with serum REG4 detection where applicable

These validation approaches enhance confidence in the specificity and reliability of REG4 antibody-based detection systems.

How should researchers interpret contradictory findings between REG4 expression and clinical outcomes?

The relationship between REG4 expression and clinical outcomes presents several complexities that require careful interpretation:

By addressing these considerations, researchers can develop more nuanced interpretations of the complex relationship between REG4 expression and clinical outcomes in cancer research.