pbpE Antibody

What is pbpE protein and what role does it play in bacterial physiology?

The pbpE protein (Penicillin-binding protein 4*) is a critical component in bacterial cell wall biosynthesis, primarily involved in peptidoglycan modification during cortex synthesis in Bacillus subtilis. This protein belongs to the penicillin-binding protein family and plays a significant role in maintaining cell wall integrity. Understanding pbpE function provides insights into bacterial growth, sporulation, and potential antimicrobial resistance mechanisms in gram-positive bacteria .

What are the validated applications for pbpE Antibody?

The pbpE Antibody (PACO50306) has been rigorously validated for multiple research applications, most notably Western Blot (WB) and ELISA. For Western Blot applications, researchers should use dilution ranges of 1:500-1:5000, while ELISA applications typically require dilutions of 1:2000-1:10000. These applications enable precise detection and quantification of pbpE protein expression across various experimental conditions .

What are the key specifications of commercially available pbpE Antibody?

The commercially available pbpE Antibody (PACO50306) is a rabbit-derived polyclonal antibody with high specificity for Bacillus subtilis samples. It is supplied in liquid form (50μg) with a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. The antibody is purified using Protein G purification (>95% purity) and is non-conjugated, making it versatile for various detection methods .

How should researchers prepare bacterial samples for optimal pbpE detection?

For optimal detection of pbpE in bacterial samples, researchers should implement a carefully designed extraction protocol:

• Culture Bacillus subtilis to mid-logarithmic phase (OD600 = 0.6-0.8) in appropriate media

• Harvest cells by centrifugation (5,000 × g for 10 minutes at 4°C)

• Wash bacterial pellet twice with ice-cold PBS to remove media components

• Resuspend in lysis buffer containing appropriate protease inhibitors

• Add lysozyme (1 mg/mL final concentration) and incubate at 37°C for 30 minutes

• Disrupt cells via sonication (6-8 cycles of 10 seconds on/20 seconds off at 30% amplitude)

• Centrifuge lysate at 15,000 × g for 30 minutes at 4°C

• Collect supernatant for soluble proteins or process pellet for membrane-associated fractions

• Determine protein concentration using Bradford or BCA assay

• Add SDS-PAGE sample buffer and heat at 95°C for 5 minutes prior to gel loading

This protocol ensures efficient extraction while preserving protein integrity for subsequent antibody-based detection methods.

What Western blot conditions yield optimal results with pbpE Antibody?

To achieve optimal Western blot results with pbpE Antibody, researchers should implement the following protocol:

• Separate 20-50 μg of protein extract on a 10% SDS-PAGE gel

• Transfer proteins to PVDF membrane (100V for 60 minutes in cold transfer buffer)

• Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Incubate with primary pbpE Antibody at 1:1000 dilution (in 5% BSA/TBST) overnight at 4°C

• Wash membrane 3× with TBST (10 minutes each)

• Incubate with HRP-conjugated anti-rabbit IgG secondary antibody (1:50000 dilution) for 1 hour

• Wash membrane 3× with TBST (10 minutes each)

• Develop using ECL substrate and image using appropriate detection system

Western blot validation data shows that the antibody detects a specific band at the expected size of 52 kDa in recombinant protein samples, confirming its specificity and sensitivity .

What controls are essential when using pbpE Antibody in immunoassays?

When designing experiments with pbpE Antibody, researchers should incorporate multiple controls to ensure data reliability:

• Positive control: Wild-type Bacillus subtilis known to express pbpE

• Negative control: Ideally a pbpE knockout strain (if available)

• Recombinant protein control: Purified recombinant pbpE protein

• Primary antibody omission control: Sample processed without primary antibody

• Isotype control: Non-specific rabbit IgG at equivalent concentration

• Blocking peptide control: Pre-incubation of antibody with immunizing peptide

• Cross-reactivity controls: Related bacterial species to assess specificity

These controls help distinguish specific signals from background noise and validate experimental findings. The inclusion of multiple controls is particularly important given ongoing concerns about antibody reproducibility in scientific research .

How can pbpE Antibody be utilized to study antimicrobial resistance mechanisms?

The pbpE Antibody provides a valuable tool for investigating antimicrobial resistance mechanisms, particularly those involving cell wall synthesis modifications. Researchers can design experiments to:

• Compare pbpE expression levels in antibiotic-sensitive versus resistant strains

• Monitor changes in pbpE expression following exposure to sub-lethal concentrations of β-lactam antibiotics

• Analyze potential structural modifications of pbpE through immunoprecipitation followed by mass spectrometry

• Perform co-localization studies with other penicillin-binding proteins to identify compensatory mechanisms

• Evaluate pbpE expression in clinical isolates with varying antibiotic susceptibility profiles

This approach provides insights into the molecular mechanisms underlying bacterial adaptation to antimicrobial challenges, potentially revealing novel therapeutic targets .

What considerations should researchers address when designing PBPK models involving antibodies against bacterial targets?

When designing physiologically based pharmacokinetic (PBPK) models involving antibodies against bacterial targets like pbpE, researchers should consider multiple factors:

• Integrate antibody-specific physiochemical properties that influence distribution and clearance

• Account for nonspecific binding parameters, which significantly correlate with vascular to endothelial clearance (R = 0.64, p = .0013)

• Consider paracellular transport mechanisms influenced by antibody characteristics (R = 0.52, p = .012)

• Incorporate thermal stability, hydrophobicity, and self-association parameters, though these alone may not be predictive

• Develop mechanism-based models rather than empirical correlations to better predict antibody pharmacokinetics

• Account for FcRn-mediated recycling that significantly impacts antibody half-life

These considerations enable more accurate prediction of antibody distribution and efficacy when targeting bacterial antigens in complex physiological systems .

How can researchers evaluate potential cross-reactivity of pbpE Antibody with homologous proteins?

Evaluating cross-reactivity is essential for accurate interpretation of experimental results. Researchers should:

• Perform sequence alignment analysis of the immunogen (Bacillus subtilis pbpE residues 1-451) against homologous proteins from relevant bacterial species

• Test the antibody against protein extracts from multiple bacterial species using Western blot

• Quantify relative signal intensity across species to assess degree of cross-reactivity

• Conduct epitope mapping to identify specific binding regions

• Validate findings using knockout strains or heterologous expression systems

• Consider pre-adsorption with recombinant proteins to enhance specificity in mixed-culture experiments

What strategies can resolve weak or absent signal when using pbpE Antibody?

When encountering weak or absent signals with pbpE Antibody, researchers should systematically evaluate:

• Protein extraction efficiency: Ensure complete lysis of bacterial cells with appropriate cell wall digestion steps

• Protein degradation: Verify protease inhibitor effectiveness and maintain samples at 4°C throughout processing

• Antibody concentration: Consider using higher antibody concentrations (1:500 dilution) for low-abundance targets

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C

• Detection sensitivity: Utilize enhanced chemiluminescence substrates for improved signal amplification

• Membrane selection: PVDF membranes often provide better protein retention than nitrocellulose

• Expression levels: Confirm pbpE expression under experimental conditions via RT-qPCR

• Antibody quality: Verify antibody activity using positive control samples

Implementing these approaches systematically helps identify and address the specific cause of signal issues .

How should researchers validate antibody specificity in the context of increasing concerns about reproducibility?

Given the significant concerns about antibody reproducibility in scientific research, rigorous validation is essential:

• Multi-method validation: Confirm specificity using orthogonal techniques (Western blot, ELISA, immunoprecipitation)

• Genetic validation: Compare results between wild-type and gene knockout samples

• Peptide competition: Demonstrate signal reduction following antibody pre-incubation with immunizing peptide

• Recombinant protein standards: Include concentration gradients of recombinant protein as quantitative references

• Lot-to-lot consistency: Test multiple antibody lots to ensure reproducible performance

• Independent verification: Consider using multiple antibodies targeting different epitopes of the same protein

• Publication of validation data: Document and share comprehensive validation methods and results

This thorough approach addresses the estimated 50% failure rate of commercial antibodies to meet basic characterization standards, which contributes to significant financial losses and reproducibility challenges in biomedical research .

What factors influence long-term stability and performance of pbpE Antibody?

To maintain optimal pbpE Antibody performance over time, researchers should consider these stability factors:

• Storage temperature: Maintain at -20°C for long-term storage with minimal freeze-thaw cycles

• Aliquoting strategy: Prepare single-use aliquots to prevent repeated freeze-thaw cycles

• Buffer composition: The provided buffer (50% Glycerol, 0.01M PBS, pH 7.4) with 0.03% Proclin 300 preservative optimizes stability

• Dilution stability: Working dilutions should be prepared fresh or stored at 4°C for no more than 1-2 weeks

• Contamination prevention: Use sterile technique when handling antibody solutions

• Temperature fluctuations: Avoid extended periods at room temperature

• Expiration monitoring: Periodically test antibody performance against reference samples

Implementing these practices ensures consistent antibody performance throughout the research timeline and enhances experimental reproducibility .

How might pbpE Antibody contribute to high-throughput screening approaches for novel antimicrobials?

The pbpE Antibody offers potential for developing high-throughput screening platforms to identify novel antimicrobial compounds:

• Develop ELISA-based assays to quantify pbpE binding following compound treatment

• Create fluorescence-based cellular assays to monitor pbpE localization changes in response to antimicrobial candidates

• Establish multiplexed detection systems combining pbpE with other penicillin-binding proteins

• Implement automated image analysis workflows for rapid assessment of pbpE expression patterns

• Develop reporter systems that correlate pbpE binding with cellular viability markers

These approaches could accelerate identification of compounds that disrupt bacterial cell wall synthesis through novel mechanisms, potentially addressing antimicrobial resistance challenges .

What considerations should researchers address when integrating pbpE Antibody into systems biology approaches?

When incorporating pbpE Antibody into systems biology investigations, researchers should:

• Validate quantitative relationships between antibody signal and actual protein concentration

• Establish standardized protocols for comparative analysis across multiple experimental conditions

• Develop data normalization approaches that account for technical variations in antibody performance

• Consider multiplexed detection methods to analyze pbpE in context with interacting proteins

• Integrate antibody-based detection data with transcriptomic and metabolomic datasets

• Implement computational models that incorporate protein-level data into pathway analysis

This integrated approach enables more comprehensive understanding of pbpE's role within broader bacterial physiological networks and stress responses .