The btfP Antibody (PACO53802) is a polyclonal antibody developed for research applications targeting the Fragilysin protein (encoded by the btfP gene) in Bacteroides fragilis. This antibody is engineered to detect and analyze Fragilysin, a diarrheal toxin with gelatinase, azocollase, and fibrinogenase activities . It is primarily used in immunological and microbiological studies to explore the role of Fragilysin in pathogenesis, immune modulation, and cellular processes .
3.1 Immunological Studies
The btfP Antibody is validated for ELISA and Western blot (WB) assays, with recommended dilutions of 1:2000–1:10,000 (ELISA) and 1:500–1:5000 (WB) . It detects a 58 kDa band corresponding to Fragilysin, enabling researchers to study:
Fragilysin-mediated pathogenesis: Its role in disrupting host tissues and immune evasion .
Transcriptional regulation: BTFP’s involvement in gene expression via BTB/POZ domain interactions .
3.2 Microbiological Analysis
The antibody aids in identifying Bacteroides fragilis isolates in clinical samples, contributing to studies on gut microbiota composition and antibiotic resistance .
Fragilysin, the target of this antibody, hydrolyzes structural proteins (e.g., actin, tropomyosin), facilitating bacterial invasion and tissue damage . Studies using this antibody have elucidated its role in:
Inflammatory bowel disease (IBD): Fragilysin’s contribution to mucosal barrier disruption .
Cancer progression: Dysregulation of BTFP in tumor microenvironments .
While the btfP Antibody is not therapeutic, its use in mapping Fragilysin’s epitopes aligns with broader trends in antibody engineering, such as biparatopic antibodies (bpAbs) that target multiple epitopes for enhanced specificity . Such approaches could inspire future anti-Fragilysin therapies .
KEGG: ag:AAB49835
The btfP Antibody (PACO53802) is a polyclonal antibody developed in rabbits that specifically targets BTB/POZ domain-containing transcription factor proteins. This antibody exhibits high specificity for Bacteroides fragilis samples and has been validated primarily for Western blot applications and ELISA assays . The antibody recognizes the recombinant Bacteroides fragilis Fragilysin protein (amino acids 26-405) as its immunogen, making it particularly valuable for researchers studying this bacterial species and its associated proteins .
As a research tool, btfP Antibody is instrumental in investigations focusing on transcriptional regulation mechanisms, protein-protein interactions involving BTB/POZ domains, and studies examining the role of these proteins in cellular processes. Methodologically, the antibody enables precise detection and analysis of target proteins in various experimental systems, providing researchers with a means to examine protein expression, localization, and interaction patterns.
When optimizing Western blot protocols with btfP Antibody, begin with a dilution range of 1:500-1:5000 as recommended by manufacturers . The optimal dilution should be determined empirically for your specific sample type and protein concentration. For most bacterial protein extracts, starting with a 1:1000 dilution typically yields satisfactory results.
For optimal performance, consider the following methodological approach:
Sample preparation: Use a buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl, 1% NP-40, supplemented with protease inhibitors
Protein loading: 20-50μg of total protein per lane for cell lysates
Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature
Primary antibody incubation: Dilute btfP Antibody in blocking solution and incubate overnight at 4°C
Washing: 3-5 washes with TBST, 5 minutes each
Detection system: HRP-conjugated anti-rabbit secondary antibody at 1:5000 dilution
For challenging samples with low target protein expression, extending the primary antibody incubation time to 16-18 hours and using enhanced chemiluminescence detection systems can significantly improve sensitivity.
For researchers investigating complex transcriptional networks involving BTB/POZ domain proteins, btfP Antibody can be integrated into multiplexed detection systems. This advanced application requires careful experimental design to avoid cross-reactivity and ensure specific protein detection.
A methodological approach for multiplexed protein detection using btfP Antibody includes:
Sequential immunoblotting: Strip and reprobe membranes with antibodies raised in different host species
Fluorescent multiplexing: Use spectrally distinct fluorophore-conjugated secondary antibodies
Microscopy applications: Combine btfP Antibody with other primary antibodies for co-localization studies
When designing multiplexed experiments, it's crucial to validate the specificity of btfP Antibody against your target protein through preliminary single-antibody experiments. For quantitative analysis, include appropriate loading controls and consider using internal standards for normalization across experimental conditions.
When facing contradictory results using btfP Antibody across different experimental systems, employ a systematic troubleshooting approach to identify the source of variability. This methodological framework helps resolve data inconsistencies:
Antibody validation: Confirm antibody specificity using positive and negative controls
Sample preparation assessment: Evaluate protein extraction methods for potential degradation
Cross-reactivity analysis: Test for potential cross-reactivity with similar protein domains
Technical replication: Perform at least three independent experiments under identical conditions
Biological variation consideration: Account for natural variation in protein expression
A comprehensive validation protocol should include knockout or knockdown controls to confirm antibody specificity. For Bacteroides fragilis experiments, comparison of wild-type and fragilysin-deficient strains can provide definitive evidence of antibody specificity .
Data from three independent experiments should be statistically analyzed to determine if observed variations fall within expected biological ranges or indicate technical issues requiring further optimization.
While btfP Antibody is primarily validated for Western blot applications, researchers adapting it for immunofluorescence should carefully optimize fixation and permeabilization protocols. The methodological approach should be tailored based on cellular localization of the target protein:
For cytoplasmic proteins: 4% paraformaldehyde fixation (10 minutes) followed by 0.1% Triton X-100 permeabilization (5 minutes)
For membrane-associated proteins: 2% paraformaldehyde fixation (15 minutes) with 0.05% saponin permeabilization
For nuclear proteins: Methanol:acetone (1:1) fixation/permeabilization (-20°C, 10 minutes)
Each fixation method preserves different epitopes, potentially affecting antibody binding. Therefore, optimization experiments comparing multiple fixation methods are recommended when adapting btfP Antibody for immunofluorescence applications. Include appropriate controls for autofluorescence and non-specific binding, particularly when working with bacterial samples that may exhibit high background fluorescence.
For researchers working with low-abundance targets, implementing enrichment techniques can significantly improve detection sensitivity when using btfP Antibody. Consider the following methodological approaches:
Immunoprecipitation pre-enrichment: Use btfP Antibody coupled to protein A/G beads for target enrichment prior to Western blot analysis
Subcellular fractionation: Concentrate target proteins through organelle isolation based on predicted localization
Sequential extraction: Use increasingly stringent buffers to extract proteins based on solubility characteristics
For bacterial protein studies, differential centrifugation to separate membrane, cytoplasmic, and extracellular fractions can significantly enrich target proteins. This approach is particularly valuable when studying secreted forms of fragilysin from Bacteroides fragilis cultures .
When analyzing enriched samples, it's crucial to normalize loading based on fraction-specific markers rather than total protein content, as enrichment procedures can distort standard protein quantification methods.
Establishing reliable quantification standards for Western blot analysis using btfP Antibody requires a systematic approach to ensure reproducibility and accuracy. The following methodological framework provides guidance for quantitative analysis:
Standard curve generation: Include a dilution series of recombinant target protein
Multiple internal controls: Use housekeeping proteins appropriate for your experimental system
Technical replication: Perform at least three independent Western blots
Densitometric analysis: Use software with background subtraction capabilities
| Sample Type | Recommended Loading Range (μg) | Optimal Antibody Dilution | Suggested Housekeeping Control |
|---|---|---|---|
| Bacterial lysates | 20-50 | 1:1000 | RNA polymerase sigma factor |
| Purified recombinant protein | 0.1-1.0 | 1:5000 | N/A (direct quantification) |
| Enriched immunoprecipitates | 10-20 | 1:2000 | IgG heavy chain |
When analyzing data, apply appropriate statistical methods to determine significance between experimental conditions. For time-course experiments, normalization to baseline expression levels often provides more meaningful comparisons than absolute values.
Distinguishing specific from non-specific signals is crucial for accurate data interpretation when using btfP Antibody. Implement this methodological approach to enhance signal specificity:
Control inclusions: Run parallel samples with pre-immune serum or IgG isotype controls
Blocking peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity
Molecular weight verification: Compare observed band sizes with predicted molecular weights
Signal-to-noise ratio analysis: Calculate and establish minimum acceptable ratios
For Western blot applications, btfP Antibody should detect the Bacteroides fragilis fragilysin protein at approximately 45-48 kDa . Bands at significantly different molecular weights should be scrutinized for potential cross-reactivity or degradation products.
When confronted with multiple bands, peptide competition assays can definitively identify specific signals, as pre-incubation with the immunizing peptide should eliminate or significantly reduce the intensity of specific bands while leaving non-specific signals largely unchanged.
For researchers investigating host-pathogen interactions, btfP Antibody serves as a valuable tool for tracking Bacteroides fragilis fragilysin expression and localization during infection. This methodological approach optimizes btfP Antibody use in infection models:
Temporal expression analysis: Monitor fragilysin expression at defined time points post-infection
Subcellular localization: Track protein distribution in bacterial and host cells using fractionation techniques
Co-immunoprecipitation: Identify host proteins interacting with fragilysin during infection
Ex vivo tissue analysis: Detect fragilysin in infected tissue samples
When designing infection experiments, include appropriate controls such as fragilysin-deficient bacterial strains and uninfected host cells. For in vivo models, consider using fluorescently labeled secondary antibodies for immunohistochemical detection of fragilysin in tissue sections.
The ability to precisely detect fragilysin using btfP Antibody enables researchers to correlate bacterial protein expression with host inflammatory responses, tissue damage, and disease progression in models of Bacteroides fragilis infection.
When investigating transcription factor function, researchers should consider the complementary nature of antibody-based detection and molecular genetic approaches. The methodological comparison below highlights when btfP Antibody offers advantages over genetic methods:
Protein-level analysis: btfP Antibody directly detects protein expression, unlike transcript-based approaches that may not reflect post-transcriptional regulation
Post-translational modification detection: Antibody-based methods can be combined with specific PTM antibodies to study protein regulation
Protein-protein interaction studies: btfP Antibody enables co-immunoprecipitation to identify protein binding partners
Spatial resolution: Immunolocalization provides information about subcellular distribution not available from genetic approaches
For comprehensive studies, integrating both antibody-based detection and molecular genetic techniques provides the most complete understanding of transcription factor biology. While CRISPR-Cas9 gene editing offers powerful tools for functional studies, btfP Antibody remains essential for confirming phenotypes at the protein level and investigating mechanistic details of protein function.
When encountering inconsistent Western blot results with btfP Antibody, implement this methodological troubleshooting framework to systematically identify and resolve issues:
Antibody validation: Confirm antibody activity using positive control samples
Sample preparation assessment: Evaluate protein extraction methods and storage conditions
Transfer efficiency verification: Use reversible staining to confirm protein transfer
Blocking optimization: Test alternative blocking agents (BSA vs. milk) for reduced background
Signal development analysis: Compare different detection methods for optimal signal-to-noise ratio
| Common Issue | Potential Cause | Methodological Solution |
|---|---|---|
| No signal | Inactive antibody or low target abundance | Increase antibody concentration; use enrichment techniques |
| Multiple bands | Cross-reactivity or protein degradation | Optimize extraction buffers with protease inhibitors; perform peptide competition |
| High background | Insufficient blocking or washing | Increase blocking time; add 0.05% Tween-20 to antibody diluent |
| Variable signal intensity | Inconsistent loading or transfer | Normalize to housekeeping proteins; standardize protein quantification methods |
Document all optimization steps and maintain a laboratory record of successful protocols to ensure reproducibility across experiments and between researchers.
Validating antibody specificity across different experimental systems is critical for ensuring reliable and reproducible results. Implement this comprehensive validation approach for btfP Antibody:
Genetic validation: Test antibody against knockout/knockdown systems where the target protein is absent
Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding
Cross-species reactivity assessment: Test against related species to determine conservation of recognition
Multiple detection methods: Confirm specificity using orthogonal techniques (e.g., mass spectrometry)
For bacterial systems, comparing wild-type Bacteroides fragilis with fragilysin-deficient mutants provides definitive validation of antibody specificity . For heterologous expression systems, comparing untransfected cells with those expressing the target protein can establish specificity in mammalian contexts.
Maintain detailed records of validation experiments, including positive and negative controls, to support the robustness of research findings and facilitate troubleshooting if specificity issues arise in future experiments.
Integrating btfP Antibody with advanced microscopy enables spatial proteomics applications that reveal the subcellular distribution and dynamics of target proteins. This methodological framework outlines approaches for combining btfP Antibody with cutting-edge microscopy techniques:
Super-resolution microscopy: Use fluorophore-conjugated secondary antibodies compatible with STORM, PALM, or STED microscopy
Live-cell imaging: Combine with genetically encoded tags for correlative light-electron microscopy
Expansion microscopy: Adapt immunolabeling protocols for physically expanded specimens
Tissue clearing techniques: Optimize penetration for whole-tissue imaging
When adapting btfP Antibody for these applications, preliminary optimization experiments should establish ideal fixation conditions, antibody concentrations, and incubation times specific to each microscopy technique. For bacterial cells, consider cell wall permeabilization methods that maintain cellular ultrastructure while allowing antibody access to intracellular targets.
The integration of btfP Antibody with advanced imaging approaches offers unprecedented insights into protein localization patterns that cannot be resolved by conventional microscopy, particularly valuable for studying bacterial protein distribution during host-pathogen interactions.
Recent advances in AI-driven protein design offer promising avenues for developing enhanced versions of btfP antibodies with improved specificity and functionality. This methodological exploration examines the potential applications of computational design approaches:
Epitope-focused optimization: Use structural prediction algorithms to design antibodies targeting highly specific epitopes
Affinity maturation: Apply computational approaches to enhance binding affinity through targeted mutations
Cross-reactivity reduction: Identify and eliminate potential cross-reactive regions in the antibody sequence
Format diversification: Design alternative antibody formats (Fab, scFv) optimized for specific applications
Recent developments in RFdiffusion technology for antibody design demonstrate the feasibility of generating novel antibodies with precisely engineered binding properties . This approach could be applied to create next-generation btfP antibodies with enhanced specificity for particular epitopes or post-translational modifications.
As these computational approaches mature, researchers can expect increasingly sophisticated antibody tools that combine high specificity with application-optimized properties, potentially revolutionizing how we detect and study bacterial proteins in complex biological systems.