RBP4 Antibody

What is RBP4 and what is its biological significance?

RBP4 is a member of the lipocalin family and serves as the major transport protein for retinol (vitamin A) in the circulatory system . In humans, RBP4 is a secreted protein with 201 amino acid residues and a molecular mass of approximately 23 kDa . It plays a crucial role in delivering retinol from liver stores to peripheral tissues, with its structure specifically designed to fit into binding pockets that facilitate this transport .

The protein is primarily expressed in blood plasma and urine, and has been associated with several pathological conditions, including retinal dystrophy, iris coloboma, and comedogenic acne syndrome . RBP4 transfers bound all-trans retinol to STRA6, which then facilitates retinol transport across cell membranes .

What are the common applications for RBP4 antibodies in research?

RBP4 antibodies are utilized in various immunodetection techniques with Western Blot (WB) being the most widely implemented application . Other common applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunohistochemistry (IHC) on paraffin-embedded tissues (IHC-p)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Immunoprecipitation (IP)

These techniques allow researchers to detect, quantify, and visualize RBP4 in various sample types, including tissue lysates, serum samples, and cultured cells.

Which species reactivity is available for RBP4 antibodies?

RBP4 antibodies exhibit cross-reactivity with multiple species due to the conserved nature of the protein. Common species reactivity includes:

Many antibodies show multi-species reactivity, with human, mouse, and rat being the most commonly targeted species for research applications .

What criteria should be considered when selecting between monoclonal and polyclonal RBP4 antibodies?

The choice between monoclonal and polyclonal RBP4 antibodies depends on the specific research application and experimental requirements:

Monoclonal Antibodies:

• Offer higher specificity by recognizing a single epitope on RBP4

• Provide consistent lot-to-lot reproducibility

• Ideal for applications requiring high specificity such as distinguishing between closely related protein isoforms

• Examples include clone EP3657 and RBP4/4046, which have been cited in multiple publications

Polyclonal Antibodies:

• Recognize multiple epitopes on RBP4, potentially providing stronger signals

• Better for detecting denatured proteins in Western blotting

• May offer greater flexibility in different applications

• Often produced using recombinant full-length human RBP4 protein as immunogen

For quantitative studies examining RBP4 expression levels in metabolic disorders or cancer research, monoclonal antibodies may provide more consistent results across experiments due to their specificity and reproducibility .

How can researchers validate RBP4 antibodies for their specific applications?

Proper validation of RBP4 antibodies is crucial for ensuring experimental reliability:

• Positive Control Testing: Use recombinant human RBP4 protein to verify antibody recognition

• Western Blot Validation: Confirm specificity by checking for a single band at the expected molecular weight (23 kDa for human RBP4)

• Cross-Reactivity Assessment: Test antibody against serum samples and liver lysates to ensure recognition of native protein

• Knockout/Knockdown Controls: Where possible, use RBP4-deficient samples as negative controls

• Comparison with Known Expression Patterns: Verify tissue distribution matches established RBP4 expression patterns (high in liver, present in blood plasma)

Researchers have successfully validated anti-RBP4 monoclonal antibodies using hybridoma technology, confirming both high affinity and specificity for binding to natural RBP4 protein .

How can RBP4 antibodies be utilized in studying metabolic disorders, particularly insulin resistance?

The relationship between RBP4 and insulin resistance remains controversial within the scientific community . When designing experiments to investigate this relationship, researchers should consider:

• Quantitative Analysis: Measure serum RBP4 levels using validated ELISA or Western blot techniques with appropriate controls

• Tissue-Specific Expression: Examine RBP4 expression in adipose tissue versus liver, as there are conflicting reports regarding the primary source of elevated RBP4 in metabolic disorders

• Functional Studies: Use RBP4 antibodies in neutralization experiments to assess the direct impact of RBP4 on insulin signaling pathways

• Species Considerations: Note that human and murine RBP4 may have different effects, as studies have shown disparate results when using human versus mouse RBP4 protein in transgenic models

Researchers should be aware that while many studies have found elevated circulating RBP4 in insulin resistance and type 2 diabetes, some have failed to reproduce improved insulin sensitivity in RBP4-deficient mice on high-fat diets . Additionally, kidney function may be a major determinant of serum RBP4 levels, which complicates interpretation in diabetic conditions .

What role does RBP4 play in liver diseases and how can antibodies help investigate this connection?

RBP4 expression levels are closely related to liver damage and may serve as important biomarkers in hepatic disorders:

• Hepatocellular Carcinoma (HCC) Studies: Immunohistochemical analysis using anti-RBP4 monoclonal antibodies has revealed that low expression of RBP4 in HCC tissues correlates with cancer development

• Biomarker Development: Transcriptomic and proteomic analyses have demonstrated reduced RBP4 expression in serum of HCC patients, suggesting potential as a diagnostic marker

• Mechanistic Investigations: RBP4 antibodies can be used to investigate the molecular mechanisms connecting RBP4 downregulation to cancer progression

• Differential Expression Analysis: Compare RBP4 levels in tumor versus adjacent non-tumor tissues using immunohistochemistry to establish expression patterns relevant to pathogenesis

For liver-focused studies, researchers should particularly consider antibodies validated for immunohistochemical applications that have demonstrated specificity in liver tissues .

What are common issues when using RBP4 antibodies in Western blotting and how can they be resolved?

When performing Western blot using RBP4 antibodies, researchers may encounter several challenges:

• Multiple Bands: If observing bands other than the expected 23 kDa band:

  • Verify sample preparation conditions (reducing vs. non-reducing)

  • Check for post-translational modifications that might alter migration patterns

  • Consider using more specific monoclonal antibodies that target unique epitopes

• Weak Signal:

  • Optimize antibody concentration (typical working concentrations range from 1-2 μg/mL for Western blot applications)

  • Increase protein loading for samples with low RBP4 expression

  • Consider longer exposure times or more sensitive detection methods

• High Background:

  • Increase blocking time or concentration

  • Optimize washing steps

  • Reduce primary antibody concentration

  • Use fresh, properly stored antibody

The successful detection of RBP4 in human serum and liver lysates has been demonstrated with antibody concentrations of 1 μg/mL, while recombinant human RBP4 protein detection may require slightly higher concentrations (2 μg/mL) .

How should RBP4 antibodies be used in immunohistochemistry of liver and adipose tissues?

For optimal immunohistochemical detection of RBP4 in tissues relevant to metabolic and liver disorders:

• Tissue Preparation:

  • Use appropriately fixed (typically formalin) and paraffin-embedded tissues

  • Consider antigen retrieval methods to expose epitopes potentially masked during fixation

• Antibody Selection:

  • Choose antibodies specifically validated for IHC-p applications

  • Consider clones that have been successfully used in published studies of liver or adipose tissues

• Protocol Optimization:

  • Titrate antibody concentration to achieve optimal signal-to-noise ratio

  • Include proper positive controls (normal liver tissue) and negative controls

  • Use detection systems appropriate for the expected expression level

• Interpretation:

  • Compare staining patterns between diseased and normal tissues

  • Note subcellular localization (RBP4 is primarily secreted but may show cytoplasmic staining in producing cells)

  • Consider dual staining with other markers to identify cell types expressing RBP4

Anti-RBP4 monoclonal antibodies developed using hybridoma technology have been successfully employed for immunohistochemical analysis of HCC and adjacent tissues, demonstrating their utility in cancer research applications .

What are unresolved questions about RBP4 that antibody-based research might help address?

Several important questions remain in RBP4 research that could benefit from advanced antibody applications:

• Post-Translational Modifications: Are there unidentified post-translational modifications of RBP4 that occur upon extended serum half-life that could mediate its effects in disease states?

• Retinol Transport Role: Is retinol transport just a bystander or an active participant in RBP4's presumed effects on metabolism and disease?

• Species-Specific Effects: Why are some metabolic effects of transgenically elevated RBP4 in mice seen with human but not murine protein?

• Inflammatory Mechanism: From a physiological perspective, how does RBP4 (normally circulating at high levels) become an inflammatory stressor upon the 2-3 fold elevation observed in metabolic disorders?

Advanced epitope mapping using different monoclonal antibodies might help identify critical functional domains and potential sites of modification that contribute to RBP4's diverse biological effects.

How might new antibody technologies enhance RBP4 research and clinical applications?

Emerging antibody technologies offer opportunities to advance RBP4 research:

• Multiplex Immunoassays: Simultaneous detection of RBP4 alongside other metabolic markers to establish correlation patterns in complex diseases

• Single-Cell Analysis: Utilizing highly specific antibodies to investigate RBP4 expression at the single-cell level within heterogeneous tissues

• Proximity Ligation Assays: Investigating protein-protein interactions between RBP4 and potential binding partners using paired antibody approaches

• Therapeutic Applications: Development of neutralizing antibodies against RBP4 could provide therapeutic options for conditions where elevated RBP4 contributes to pathology

• Standardized Diagnostic Kits: Development of consistent, highly specific antibody-based assays for RBP4 quantification would address current issues with variability between commercial antibodies and kits from different manufacturers and batches