BPIFA4P Antibody

What is BPIFA4P and what is its role in immunology?

BPIFA4P (BPI Fold Containing Family A, Member 4, Pseudogene) is a member of the bactericidal/permeability-increasing protein (BPI) family implicated in immune defense mechanisms. This protein plays an important role in innate immunity, specifically in the defense against microbial pathogens . Research involving BPIFA4P has implications for the development of novel therapies for infectious diseases and for gaining insights into immune responses to microbial invaders . Also known as Latherin (LATH), this protein is a major component in sweat and has surfactant properties . Understanding BPIFA4P function is crucial for developing strategies to enhance immune defense mechanisms.

What applications are BPIFA4P antibodies validated for?

BPIFA4P antibodies have been validated for multiple research applications:

Most commercially available BPIFA4P antibodies are polyclonal antibodies produced in rabbits, showing high reactivity with human samples . Some antibodies also demonstrate cross-reactivity with mouse and rat samples . Researchers should verify the specific applications and species reactivity for their particular antibody before use.

What is the molecular weight discrepancy observed with BPIFA4P?

An interesting phenomenon with BPIFA4P is the significant discrepancy between its calculated and observed molecular weights. According to product information:

• Calculated molecular weight: approximately 19.5 kDa

• Observed molecular weight in SDS-PAGE/Western blot: approximately 72 kDa

How are BPIFA4P antibodies typically validated?

Proper validation of BPIFA4P antibodies typically involves multiple approaches:

• Western blot analysis: Checking for bands of the expected molecular weight (noting the discrepancy mentioned above)

• Immunohistochemistry validation: Using known positive tissues (such as human breast carcinoma tissue) and assessing the staining pattern

• Immunofluorescence validation: Testing in cell lines with known expression (e.g., HeLa cells) and evaluating subcellular localization

• Peptide blocking: Pre-absorption with the immunizing peptide should abolish specific staining, confirming antibody specificity

• Cross-reactivity testing: Assessing reactivity across multiple species and related proteins

Antibodies should demonstrate specificity, selectivity, and reproducibility in the specific context for which they will be used .

What are the best controls for BPIFA4P antibody experiments?

For rigorous BPIFA4P research, the following controls are recommended:

Positive controls:

• Tissues known to express BPIFA4P (breast tissue, salivary glands)

• Cell lines with confirmed BPIFA4P expression (HeLa cells for immunofluorescence)

• Overexpression systems where BPIFA4P has been transfected

Negative controls:

• No primary antibody control to assess secondary antibody background

• Isotype controls to identify non-specific binding

• Ideally, BPIFA4P knockout or knockdown samples (considered the gold standard for antibody specificity)

• Peptide competition/blocking experiments using the immunizing peptide

The most stringent validation uses orthogonal methods to confirm findings, comparing antibody-based detection with RNA expression data or alternative detection methods .

How can I optimize BPIFA4P antibody usage for detection in tissues with low expression?

For tissues with low BPIFA4P expression, several optimization strategies can improve detection:

• Signal amplification: Employ tyramide signal amplification (TSA) or polymer detection systems to enhance sensitivity

• Antigen retrieval optimization: Test multiple antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0)

  • HIER with EDTA buffer (pH 9.0)

  • Enzymatic retrieval with proteinase K for certain epitopes

• Antibody concentration: For IHC applications, start with dilutions at the lower end of the recommended range (1:50-1:100) and titrate to optimize signal-to-noise ratio

• Incubation conditions: Extended primary antibody incubation (overnight at 4°C) often improves detection of low-abundance targets

• Detection system selection: For fluorescence applications, select brightest available fluorophores and optimize microscope settings

Systematic optimization of these parameters can significantly improve detection of low-abundance BPIFA4P in challenging samples.

How can I troubleshoot inconsistent results with BPIFA4P antibodies?

When encountering inconsistent results with BPIFA4P antibodies, consider:

• Application-specific factors:

  • For IHC/IF: Fixation methods, antigen retrieval conditions, and blocking reagents can dramatically affect epitope accessibility

  • For Western blot: Protein extraction methods, denaturation conditions, and transfer parameters impact detection

• Epitope considerations:

  • The immunizing peptide location affects antibody performance in different applications

  • BPIFA4P antibodies are often raised against specific amino acid ranges (e.g., 71-120)

  • Post-translational modifications may mask epitopes

• Technical parameters:

  • Antibody concentration should be independently optimized for each application

  • Buffer composition affects antibody binding efficiency

  • Lot-to-lot variation may require revalidation with new antibody lots

• Sample preparation standardization:

  • Consistent fixation times for tissues

  • Standardized protein extraction protocols

  • Consistent blocking conditions

Systematic documentation of all experimental conditions and changing only one variable at a time will help identify the source of inconsistency.

How do different fixation methods affect BPIFA4P antigenicity in immunohistochemistry?

Fixation significantly impacts BPIFA4P detection in immunohistochemistry:

For optimal BPIFA4P detection:

• Avoid over-fixation which excessively cross-links proteins

• Document fixation conditions meticulously

• Perform a fixation time-course study when establishing new protocols

• Consider dual fixation approaches for multiplex applications

Fixation optimization is particularly important when studying BPIFA4P alongside other targets in multiplex immunohistochemistry applications.

What are potential cross-reactivity concerns with BPIFA4P antibodies?

Cross-reactivity is an important consideration with BPIFA4P antibodies:

• BPI family homology: As a member of the BPI protein family, BPIFA4P shares structural domains with related proteins that may cause cross-reactivity

• Epitope specificity: Antibodies targeting conserved regions of the BPI fold are more likely to exhibit cross-reactivity compared to those targeting unique sequences

• Species considerations: Cross-reactivity profiles vary across species due to protein homology differences

Researchers should:

• Perform Western blot analysis to confirm single-band specificity at the appropriate molecular weight

• Test antibodies in samples with confirmed absence of BPIFA4P expression

• Use peptide blocking to verify specificity

• Consider orthogonal detection methods to validate findings

The antibody validation paper notes that "blocking peptides can prove that an antibody is bad... they cannot prove that an antibody is good" , highlighting the importance of comprehensive validation approaches.

What are the latest research findings regarding BPIFA4P's role in infectious disease?

Research suggests BPIFA4P plays important roles in:

• Antimicrobial defense: As a BPI family member, BPIFA4P is implicated in immune defense mechanisms against microbial pathogens

• Innate immunity: BPIFA4P contributes to first-line defenses at mucosal surfaces and may have direct antimicrobial activity

• Therapeutic potential: Understanding BPIFA4P function has implications for developing novel therapies for infectious diseases

Current research approaches include:

• Expression profiling in infectious disease models

• Functional assays to determine antimicrobial mechanisms

• Structure-function studies of BPIFA4P domains

Investigations into BPIFA4P's role in infectious disease could provide valuable insights for enhancing immune defense mechanisms and combating infections.

How can BPIFA4P antibodies be used in multiplexed immunoassays?

Multiplexed detection of BPIFA4P alongside other markers provides comprehensive biological context:

• Multiplexing methodologies:

  • Sequential multiplex IHC with antibody stripping/quenching between rounds

  • Spectral unmixing for fluorescent multiplex applications

  • Tyramide signal amplification (TSA) for sequential multiplexing with antibodies from the same species

• BPIFA4P multiplexing considerations:

  • Select antibody clones validated for multiplexing conditions

  • Optimize antibody concentration individually within the multiplex panel

  • Test for potential cross-reactivity or antibody interference

  • Determine optimal antibody sequence to preserve sensitive epitopes

• Potential multiplex panels including BPIFA4P:

  Target	Purpose	Recommended Fluorophore
  BPIFA4P	Primary target	FITC
  Immune cell markers (CD45, CD68)	Assess immune context	Cy3, AF555
  Epithelial markers (Cytokeratins)	Tissue context	Cy5
  Functional markers (Ki-67, cleaved caspase-3)	Biological activity	Cy7 (with TSA)

• Quality control requirements:

  • Include single-stained controls for spectral unmixing

  • Incorporate method-matched quantitative standards

  • Validate antibody performance in multiplexed conditions

These approaches enable comprehensive analysis of BPIFA4P in complex biological contexts, providing deeper insights into its interactions with other cellular systems.