ompF Antibody, Biotin conjugated is a rabbit polyclonal antibody specifically designed to recognize and bind to the outer membrane protein F (ompF) of Escherichia coli bacteria . The antibody is chemically modified through conjugation with biotin molecules, which significantly enhances its utility in immunological detection methods .
The target protein, ompF, is also known by several synonyms including outer membrane protein 1A, outer membrane protein B, outer membrane protein IA, and Porin OmpF . Additional gene identifiers for ompF include cmlB, coa, cry, tolF, ECK0920, and JW0912 . This protein serves critical functions in bacterial physiology, forming small water-filled channels and playing a structural role in determining cell shape and facilitating growth in low-osmolarity environments .
Biotin conjugation provides this antibody with significant advantages for detection purposes, as biotin forms extremely strong interactions with streptavidin and avidin proteins, enabling highly sensitive detection systems in various research applications .
The ompF Antibody, Biotin conjugated is produced in rabbits and belongs to the immunoglobulin G (IgG) class of antibodies . As a polyclonal antibody, it contains a heterogeneous mixture of immunoglobulins that recognize different epitopes on the target ompF protein . This diversity of binding sites offers advantages in certain detection applications where maximum coverage of the target antigen is desired.
The physical characteristics of ompF Antibody, Biotin conjugated include its liquid form, which is maintained in a specialized buffer solution to ensure stability . The antibody demonstrates high purity levels, typically exceeding 95% as determined by protein G chromatography purification methods . The following table summarizes the key physical and chemical properties:
Property | Specification |
---|---|
Physical Form | Liquid |
Purity | >95% |
Purification Method | Protein G chromatography |
Buffer Composition | 0.01M PBS, pH 7.4, 0.03% Proclin-300, 50% glycerol |
Uniprot ID of Target | P02931 |
Molecular Weight | Standard IgG (~150 kDa) plus biotin conjugation |
The ompF Antibody, Biotin conjugated finds utility in various immunological techniques, with the primary validated application being Enzyme-Linked Immunosorbent Assay (ELISA) . This application leverages the strong biotin-streptavidin interaction to create sensitive detection systems for the presence of ompF proteins.
Additional potential applications, based on similar antibody preparations, include:
Western Blotting for protein detection and size determination
Immunohistochemistry for localization studies in bacterial samples
Immunofluorescence when coupled with streptavidin-fluorophore conjugates
Flow cytometry for bacterial classification and analysis
The versatility of this biotin-conjugated antibody stems from its compatibility with various streptavidin-linked detection systems, including enzymatic, fluorescent, and nanoparticle-based reporters .
Research involving biotin-conjugated antibodies provides valuable insights into membrane protein interactions. Studies have demonstrated that the presentation of membrane-bound ligands significantly influences protein-ligand interactions . For example, when comparing biotin/anti-biotin systems with 2,4-dinitrophenyl (DNP)/anti-DNP systems, researchers found that while solution binding constants were similar, their behavior at membrane interfaces differed dramatically .
The dissociation constant for anti-biotin antibody was three orders of magnitude stronger than for anti-DNP at lipid membrane interfaces (Kᴅ = 3.6 ± 1.1 nM vs. 2.0 ± 0.2 μM) . This difference is attributed to the relative lipophilicity of the ligands, with more hydrophobic molecules like DNP interacting more strongly with lipid bilayers, reducing their availability to antibodies .
When compared to non-conjugated ompF antibodies, the biotin-conjugated version offers distinct advantages in detection sensitivity and versatility . The following table outlines the key differences:
Feature | ompF Antibody, Biotin Conjugated | Non-Conjugated ompF Antibody |
---|---|---|
Detection Method | Indirect (requires streptavidin) | Requires secondary antibody |
Signal Amplification | High (through avidin-biotin systems) | Moderate (dependent on secondary antibody) |
Versatility | Compatible with multiple detection systems | Limited by species-specific secondary antibodies |
Background Signal | Potentially lower | Variable depending on secondary antibody |
Direct Visualization | No (requires additional step) | No (requires additional step) |
The ompF antibody is available with various conjugations beyond biotin, including FITC (fluorescein isothiocyanate) and HRP (horseradish peroxidase) . Each conjugation offers specific advantages:
Conjugation Type | Key Advantages | Primary Applications |
---|---|---|
Biotin | High sensitivity, versatile detection options | ELISA, immunohistochemistry, multiple detection systems |
FITC | Direct fluorescent detection, no additional reagents needed | Flow cytometry, fluorescence microscopy |
HRP | Direct enzymatic detection, compatible with multiple substrates | Western blotting, ELISA, immunohistochemistry |
The choice between these conjugation types depends on specific research requirements, detection methods available, and experimental design considerations .
Research findings indicate that the presentation of biotin within membrane environments significantly influences antibody binding characteristics . When biotin ligands are screened by a poly(ethylene glycol) (PEG) polymer brush, the dissociation constant for anti-biotin antibody binding can be weakened by three orders of magnitude (2.4 ± 1.1 μM) .
Conversely, tethering hapten molecules to extended PEG linkers can significantly enhance antibody binding strength. For example, when DNP haptens were attached to long hydrophilic PEG lipopolymers rather than short lipid-conjugated tethers, the dissociation constant improved substantially (Kᴅ = 21 ± 10 nM) .
These findings have important implications for the use of biotin-conjugated antibodies in membrane protein research :
The membrane environment substantially alters antibody-antigen interactions compared to solution-phase binding
Ligand presentation strategies can be manipulated to enhance or reduce binding affinity
Experimental design should account for these effects when studying membrane proteins
Interpretation of binding data requires consideration of the lipid environment
For optimal results when working with ompF Antibody, Biotin conjugated, researchers should follow these handling guidelines :
Centrifuge the product briefly before opening to ensure all liquid is at the bottom of the container
Use sterile techniques when preparing dilutions
Prepare working dilutions fresh before use
Determine optimal working dilutions empirically for each application
Follow standard laboratory safety procedures for handling antibodies
Commercial preparations of ompF Antibody, Biotin conjugated typically undergo rigorous quality control testing to ensure reactivity, specificity, and conjugation efficiency . When selecting a product for research, consider:
Validated applications specified by the manufacturer
Purity level (typically >95% for research-grade antibodies)
Specificity testing methods and results
Lot-to-lot consistency information
Technical support availability for troubleshooting
KEGG: ecj:JW0912
STRING: 316385.ECDH10B_0999
OmpF (Outer membrane protein F) is a major porin found in the outer membrane of Gram-negative bacteria, particularly Escherichia coli. It forms trimeric channels that allow passive diffusion of small hydrophilic molecules across the bacterial outer membrane. Also known as Outer membrane protein 1A, Outer membrane protein B, or Porin OmpF, this protein has synonyms including cmlB, coa, cry, and tolF .
OmpF is significant in bacterial research for several reasons:
It plays a crucial role in controlling membrane permeability and is involved in antibiotic resistance mechanisms
The protein serves as an important molecular marker for bacterial identification and characterization
OmpF expression changes in response to environmental conditions, making it valuable for studying bacterial adaptation
It serves as a target for bacteriophage attachment, contributing to bacterial-phage interaction studies
The 39 kDa protein (P02931) is highly conserved among E. coli strains, making antibodies against it valuable tools for bacterial detection and research .
Polyclonal and monoclonal antibodies targeting ompF differ in several important aspects that influence their research applications:
Polyclonal ompF Antibodies:
Derived from multiple B-cell lineages in immunized animals (typically rabbits, as seen in commercially available options)
Recognize multiple epitopes on the ompF protein, providing robust detection even if some epitopes are altered
Generally offer higher sensitivity due to binding to multiple sites
May show batch-to-batch variation, requiring validation across lots
Examples include rabbit polyclonal ompF antibodies that are biotin-conjugated or unconjugated
Monoclonal ompF Antibodies:
Derived from single B-cell clones, producing antibodies recognizing a specific epitope
Exhibit high specificity but may be more vulnerable to epitope masking or modification
Show excellent consistency between batches
Available in various forms, including recombinant mouse anti-E. coli OmpF antibodies
For applications requiring detection of multiple strains or variants, polyclonal antibodies may be preferable, while monoclonal antibodies offer advantages in experiments requiring high specificity or reproducibility over extended periods .
Biotin conjugation significantly enhances the utility of ompF antibodies through several mechanisms:
Increased sensitivity: The biotin-avidin/streptavidin system provides signal amplification due to the high affinity (Kd ≈ 10^-15 M) between biotin and avidin/streptavidin, enabling detection of low-abundance ompF proteins
Versatile detection options: Biotin-conjugated antibodies can be detected using various avidin/streptavidin conjugates (HRP, fluorophores, gold particles), allowing researchers to select the optimal detection system for their specific application without requiring multiple specially-conjugated primary antibodies
Reduced background: The biotin-streptavidin interaction is highly specific, which can reduce non-specific binding compared to direct enzyme conjugation
Compatibility with multiple detection platforms: Biotin-conjugated ompF antibodies work effectively across diverse applications including ELISA, Western blotting, immunohistochemistry, and immunoprecipitation
Enhanced protein stability: Biotin conjugation typically has minimal impact on antibody function compared to direct enzyme conjugation, often resulting in longer shelf-life and consistent performance
For researchers studying ompF across multiple experimental platforms, biotin-conjugated antibodies provide flexibility and enhanced sensitivity, particularly valuable when working with limited bacterial samples or in cases requiring multiplex detection .
Sample preparation is critical for successful detection of ompF in bacterial cultures. The following methodological approaches optimize results:
For Western Blotting:
Culture preparation: Grow E. coli to mid-log phase (OD600 = 0.6-0.8) to ensure optimal ompF expression
Cell lysis: Use gentle lysis methods to preserve membrane protein integrity:
Sonication (5-10 pulses of 15 seconds each) in buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, and 1% Triton X-100
Add protease inhibitor cocktail to prevent protein degradation
Membrane fraction isolation: Centrifuge lysate at 15,000g for 30 minutes at 4°C to pellet membrane fractions
Sample preparation: Heat samples at 90-95°C for 5-10 minutes in SDS-PAGE loading buffer containing β-mercaptoethanol to denature the protein
For ELISA:
Membrane extraction: Isolate outer membrane fractions using sucrose gradient ultracentrifugation or selective detergent extraction
Protein quantification: Use Bradford or BCA assay to standardize protein amounts
Coating optimization: Dilute membrane extracts to 1-5 µg/ml in carbonate buffer (pH 9.6) for optimal plate coating
For Immunohistochemistry:
Fixation: Use 4% paraformaldehyde for 15-20 minutes to preserve bacterial morphology
Permeabilization: Treat with 0.1% Triton X-100 for 5-10 minutes to allow antibody access to membrane proteins
Blocking: Block with 5% BSA or 10% normal serum for 1 hour to reduce non-specific binding
These methodologies provide reliable detection of ompF while preserving its native structure and epitope accessibility for antibody binding .
A comprehensive Western blot protocol optimized for biotin-conjugated ompF antibody detection should follow these methodological steps:
Sample Preparation:
Prepare bacterial lysates as described in question 2.1
Load 10-20 μg of protein per lane on a 10-12% SDS-PAGE gel (optimal for resolving the 39 kDa ompF protein)
Electrophoresis and Transfer:
Run SDS-PAGE at 100-120V until adequate separation is achieved
Transfer proteins to PVDF or nitrocellulose membrane (0.45 μm pore size) at 100V for 1 hour or 30V overnight at 4°C
Verify transfer efficiency using reversible Ponceau S staining
Immunodetection:
Block membrane with 5% non-fat milk or 3% BSA in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature
Incubate with biotin-conjugated ompF antibody at 1:200-1:1000 dilution in blocking buffer overnight at 4°C
Wash 3-5 times with TBST, 5 minutes each
Incubate with streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature
Wash 3-5 times with TBST, 5 minutes each
Develop using ECL substrate and capture signal with appropriate imaging system
Controls and Validation:
Positive control: Include purified recombinant ompF protein or known ompF-expressing E. coli strain lysate
Negative control: Include lysate from bacterial strain with ompF deletion or non-expressing species
Loading control: Probe for constitutively expressed bacterial protein (e.g., RNA polymerase)
Expected Results:
A specific band at approximately 39 kDa corresponding to ompF protein
Signal intensity should correlate with ompF expression levels in samples
This protocol establishes conditions suitable for specific and sensitive detection of ompF using biotin-conjugated antibodies while minimizing potential background signal .
Optimizing ELISA protocols for quantitative analysis of ompF using biotin-conjugated antibodies requires systematic consideration of multiple parameters:
Protocol Optimization:
Parameter | Recommended Condition | Justification |
---|---|---|
Coating Buffer | Carbonate buffer (pH 9.6) | Optimal for hydrophobic membrane proteins like ompF |
Coating Concentration | 1-5 μg/ml of membrane fraction | Ensures sufficient antigen without saturation |
Blocking Solution | 3% BSA or 5% non-fat milk in PBS | Effectively reduces non-specific binding |
Primary Antibody | 1:500-1:2000 dilution of biotin-conjugated anti-ompF | Determined from titration experiments |
Detection System | Streptavidin-HRP (1:5000-1:10000) | Provides sensitive detection with low background |
Substrate | TMB with stop solution | Offers appropriate sensitivity range |
Washing | PBS with 0.05% Tween-20, 4-5 washes | Reduces background while preserving specific binding |
Quantification Approach:
Standard Curve Preparation:
Use purified recombinant ompF protein in a 2-fold dilution series (0.1-100 ng/mL)
Run in triplicate for statistical validity
Fit data to appropriate curve (typically 4-parameter logistic)
Sample Preparation Considerations:
Quality Control Metrics:
Coefficient of variation (CV) should be <15% between replicates
Signal-to-noise ratio should exceed 5:1
Z-factor >0.5 indicates excellent assay quality
Data Analysis:
This optimized ELISA protocol provides sensitive and reproducible quantification of ompF protein across various bacterial samples, facilitating comparative studies of expression under different conditions .
Non-specific binding with ompF antibodies can arise from multiple sources. Understanding these mechanisms and implementing appropriate countermeasures is essential for generating reliable data:
Common Causes and Solutions for Non-specific Binding:
Cross-reactivity with structurally similar porins:
Insufficient blocking:
Excessive antibody concentration:
Ineffective washing:
Sample protein overloading:
Secondary detection system issues:
Experimental Validation of Specificity:
Include knockout or ompF-depleted controls to confirm signal specificity
Perform peptide competition assay using the immunizing peptide to validate antibody specificity
Consider using orthogonal detection methods (e.g., mass spectrometry) to confirm target identity
Implementing these measures systematically can significantly reduce non-specific binding and ensure that observed signals accurately reflect ompF presence and quantity in experimental samples .
Weak or absent signals when using biotin-conjugated ompF antibodies can result from various factors in the experimental workflow. Systematic troubleshooting requires identifying and addressing these potential issues:
Sample-Related Issues:
Low ompF expression levels:
Protein degradation:
Epitope masking or denaturation:
Antibody-Related Issues:
Antibody deterioration:
Insufficient antibody concentration:
Poor biotin conjugation quality:
Technical Issues:
Inefficient protein transfer (Western blot):
Detection system limitations:
Buffer incompatibility:
Troubleshooting Decision Tree:
First, test positive controls (known ompF-containing sample) to verify detection system function
If positive control works, check sample preparation and protein extraction efficiency
If positive control fails, systematically test antibody dilution, fresh detection reagents, and alternative buffers
Addressing these common issues through systematic troubleshooting can significantly improve signal detection and ensure reliable results when working with biotin-conjugated ompF antibodies .
Biotin-conjugated ompF antibodies provide powerful tools for investigating bacterial membrane permeability and antibiotic resistance mechanisms through several sophisticated methodological approaches:
Quantifying OmpF Expression in Resistant Strains:
Comparative proteomics:
Flow cytometry applications:
Spatiotemporal Analysis of OmpF Distribution:
Super-resolution microscopy:
Real-time expression monitoring:
Structure-Function Analysis:
Epitope mapping:
Co-localization studies:
Combining ompF detection with other membrane components (using differentially labeled antibodies) reveals changes in membrane organization during resistance development
Multiplex imaging with biotin-conjugated ompF antibody and other fluorescent probes can map the relationship between ompF and other resistance-associated proteins
Example Experimental Design:
Research Question | Methodology | Expected Outcome |
---|---|---|
Does antibiotic exposure alter ompF expression over time? | Time-course Western blot analysis with biotin-conjugated ompF antibody following antibiotic exposure | Quantitative data showing temporal changes in ompF expression during adaptation |
Is ompF distribution altered in resistant strains? | Super-resolution microscopy using biotin-conjugated ompF antibody and fluorescent streptavidin | Visual evidence of altered clustering or localization patterns |
Do specific mutations affect antibody binding to ompF? | ELISA comparing antibody binding to wild-type vs. mutant ompF variants | Identification of structural regions critical for function |
These advanced applications demonstrate how biotin-conjugated ompF antibodies can contribute to mechanistic understanding of membrane permeability and antibiotic resistance beyond simple detection assays .
Simultaneous detection of multiple bacterial membrane proteins including ompF requires sophisticated multiplex approaches that leverage the versatility of biotin-conjugated antibodies:
Multiplex Western Blotting Strategies:
Sequential reprobing:
After detecting ompF with biotin-conjugated antibody, strip the membrane and reprobe for additional targets
Use complete stripping validation to ensure removal of previous antibodies
Advantage: Simple equipment requirements
Limitation: Potential for incomplete stripping and protein loss during stripping
Multi-color fluorescent detection:
Multiplex Immunofluorescence Microscopy:
Spectral unmixing approaches:
Use biotin-conjugated ompF antibody with spectrally distinct streptavidin conjugates alongside other antibodies with non-overlapping spectra
Apply computational spectral unmixing to separate signals
Advantage: High-resolution spatial information about multiple proteins
Sequential labeling protocols:
Multiplex ELISA and Protein Array Methods:
Bead-based multiplex assays:
Couple capture antibodies to spectrally distinct beads
Include biotin-conjugated ompF antibody in detection cocktail
Flow cytometry-based signal detection
Advantage: Quantitative data for multiple proteins simultaneously
Limitation: Requires specialized equipment and careful cross-reactivity control
Microarray approaches:
Quantitative Multiplexing Example:
Target Protein | Primary Antibody | Detection System | Emission Wavelength |
---|---|---|---|
OmpF | Biotin-conjugated anti-ompF | Streptavidin-Alexa Fluor 488 | 519 nm |
OmpC | Mouse monoclonal anti-OmpC | Anti-mouse-Alexa Fluor 594 | 617 nm |
OmpA | Rabbit polyclonal anti-OmpA | Anti-rabbit-Alexa Fluor 647 | 665 nm |
By carefully selecting compatible antibody pairs and detection systems, researchers can obtain quantitative and spatial information about multiple bacterial membrane proteins simultaneously, enabling advanced studies of membrane composition and organization in various bacterial physiological states .
Maintaining the activity of biotin-conjugated ompF antibodies requires careful attention to storage conditions and handling practices. The following evidence-based guidelines maximize antibody shelf life and performance:
Short-term Storage (up to 2 weeks):
Store at 2-8°C (refrigeration) in the original container
Avoid exposure to direct light
Keep container tightly closed to prevent evaporation
Do not add preservatives beyond those already present in the formulation
Long-term Storage:
Store at -20°C to -80°C in small working aliquots (typically 10-20 μL) to avoid repeated freeze-thaw cycles
Use glycerol-containing buffer (typically 50% glycerol) as a cryoprotectant
Ensure complete thawing before use and gentle mixing (avoid vortexing)
Storage Buffer Considerations:
Optimal buffer components: 10 mM PBS, pH 7.4, 0.02% sodium azide, 50% glycerol
The presence of preservatives like 0.03% Proclin 300 helps prevent microbial contamination
Avoid buffers containing detergents for long-term storage as they may gradually denature antibodies
Critical Stability Parameters:
Parameter | Recommendation | Rationale |
---|---|---|
Storage Temperature | -20°C to -80°C long-term | Minimizes protein degradation and biotin-streptavidin binding capacity loss |
Freeze-Thaw Cycles | Limit to ≤5 cycles | Each cycle can reduce activity by 5-15% |
Light Exposure | Protect from light | Prevents photobleaching of biotin and fluorescent contaminants |
Concentration | Store at ≥0.5 mg/mL | Higher concentrations generally provide better stability |
pH Stability | Maintain pH 7.2-7.6 | Optimal for IgG stability |
Documentation Practices:
Record receipt date, lot number, and initial concentration
Document each freeze-thaw cycle and volume removed
Track expiration date (typically 12 months from reconstitution)
Adherence to these storage guidelines maximizes the functional lifespan of biotin-conjugated ompF antibodies, ensuring consistent experimental results and reducing the need for frequent replacement of these valuable reagents .
Comprehensive validation of ompF antibodies before experimental use is essential to ensure reliable and reproducible results. The following methodological approach provides a systematic framework for antibody validation:
1. Positive and Negative Control Testing:
Positive controls: Test antibody against purified recombinant ompF protein or lysates from wild-type E. coli known to express ompF
Negative controls: Evaluate reactivity against:
2. Western Blot Validation:
Run side-by-side comparisons of positive and negative controls
Confirm detection of a single band at the expected molecular weight (39 kDa)
Evaluate signal-to-noise ratio and background levels
Perform dilution series to determine optimal working concentration
3. Peptide Competition Assay:
Pre-incubate antibody with excess immunizing peptide (10-100 fold molar excess)
Compare signal between blocked and unblocked antibody samples
Specific binding should be significantly reduced in the peptide-blocked condition
4. Cross-Reactivity Assessment:
Test against closely related porins (OmpC, OmpA) to evaluate specificity
Perform dot blot analysis with purified proteins of similar structure
5. Application-Specific Validation:
For each intended application (WB, ELISA, IHC), perform:
6. Lot-to-Lot Comparison (when changing lots):
Run parallel experiments with previous and new antibody lots
Quantitatively compare signal intensity, background, and specificity
Document any variations that may require protocol adjustments
Documentation Template for Antibody Validation:
Validation Parameter | Methods Used | Acceptance Criteria | Results |
---|---|---|---|
Target Specificity | Western blot against +/- controls | Single band at 39 kDa, absent in negative control | Pass/Fail |
Peptide Competition | Pre-incubation with immunizing peptide | >80% signal reduction | Pass/Fail |
Cross-Reactivity | Testing against related porins | <10% signal vs. ompF | Pass/Fail |
Application Performance | Titration in intended application | Signal:noise >5:1 at optimal dilution | Pass/Fail |
Lot Consistency | Comparison with previous lot | Variation <20% in signal intensity | Pass/Fail |
This systematic validation approach ensures that biotin-conjugated ompF antibodies meet the performance requirements for specific experimental applications, reducing the risk of generating misleading or irreproducible results .
By implementing these comprehensive validation protocols, researchers can confidently proceed with experimental applications using biotin-conjugated ompF antibodies that have demonstrated appropriate specificity and performance characteristics.