fkpA Antibody, FITC conjugated

What is fkpA Antibody, FITC conjugated and what is its target?

fkpA Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against the FKBP-type peptidyl-prolyl cis-trans isomerase FkpA protein. The antibody specifically targets the FkpA protein, which is also known as PPIase (EC 5.2.1.8) or Rotamase. The antibody is conjugated to Fluorescein isothiocyanate (FITC), allowing for fluorescent detection in various applications. The immunogen typically used is recombinant Escherichia coli O6:H1 FKBP-type peptidyl-prolyl cis-trans isomerase FkpA protein, specifically amino acids 26-270 . This antibody exhibits reactivity against Escherichia coli O6:H1, making it a valuable tool for studying this bacterial protein in research settings.

What is the biological function of FkpA protein?

FkpA functions as a peptidyl-prolyl cis-trans isomerase (PPIase) that catalyzes the rate-limiting protein folding step at peptidyl bonds preceding proline residues. PPIases like FkpA accelerate the folding of proteins by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . FkpA has a dual function: it exhibits chaperone activity through its mainly α-helical N-domain and peptidyl-prolyl-cis-trans-isomerase activity via its anti-parallel β-pleated sheet C-domain . The protein is synthesized in response to extracytoplasmic stress and assists in the assembly of outer membrane proteins while preventing aggregation of misfolded periplasmic protein derivatives . Recent research has demonstrated that FkpA is essential for imported colicin M toxicity in bacterial systems, highlighting its importance beyond just protein folding .

What are the optimal storage conditions for fkpA Antibody, FITC conjugated?

The optimal storage conditions for fkpA Antibody, FITC conjugated are:

For maximum stability and activity retention, it is crucial to aliquot the antibody upon receipt to minimize freeze-thaw cycles . The preservative (Proclin 300) and high glycerol content help maintain antibody stability during storage. When handling the antibody, always use sterile technique and avoid contamination, which could reduce shelf life and performance in experimental applications.

What applications has the fkpA Antibody, FITC conjugated been validated for?

The fkpA Antibody, FITC conjugated has been validated for several research applications:

The antibody has been definitively validated for ELISA applications across multiple sources . Due to its FITC conjugation, the antibody is theoretically suitable for applications requiring fluorescent detection, such as immunofluorescence and flow cytometry, though specific validations for these applications are not explicitly mentioned in the available data. Researchers should perform their own validation when using this antibody in applications beyond ELISA, particularly when working with different sample types or experimental conditions .

How does the structure of FkpA relate to its dual chaperone and PPIase functions?

FkpA exhibits a distinct structural organization that directly relates to its dual functionality. The protein consists of two domains with different structural characteristics:

• N-terminal Domain (α-helical): This domain predominantly contains α-helical structures and is responsible for FkpA's chaperone function. The chaperone activity enables FkpA to assist in the proper folding of newly synthesized proteins and prevent aggregation of misfolded proteins, independent of its isomerase activity .

• C-terminal Domain (β-pleated sheet): This domain features an anti-parallel β-pleated sheet structure that confers the peptidyl-prolyl-cis-trans-isomerase (PPIase) activity. This domain catalyzes the isomerization of peptide bonds preceding proline residues, a rate-limiting step in protein folding .

The dual-domain structure allows FkpA to function both as a traditional PPIase and as a molecular chaperone. Experimental evidence shows that the PPIase activity can be specifically inhibited by the immunosuppressant FK506, which binds to the C-terminal domain . In vitro studies have demonstrated that purified FkpA-His increases RNase T1 refolding 27-fold over spontaneous refolding at a concentration of 35 nM, and this activity is completely inhibited by 12 μM FK506 . This structural arrangement makes FkpA particularly effective in assisting protein folding under stress conditions in the bacterial periplasm.

What is the relationship between FkpA and colicin M toxicity in bacterial systems?

The relationship between FkpA and colicin M toxicity represents a novel biological function of this periplasmic chaperone. Research has revealed that:

• FkpA is essential for the toxicity of imported colicin M in bacterial systems. Mutants in the fkpA gene display specific resistance to high colicin M concentrations, while no other tested chaperone mutants conferred similar resistance .

• The tolM gene, previously associated with colicin M resistance, has been identified as identical to fkpA. This finding establishes a direct genetic link between FkpA and colicin M sensitivity .

• Complementation studies have shown that mutant strains regain sensitivity to colicin M when transformed with wild-type fkpA, confirming the causal relationship .

• Temperature sensitivity was observed in the complementation experiments. The temperature-sensitive mutant K458 complemented with temperature-sensitive FkpA (FkpA43-His) was sensitive to colicin M at 27°C but insensitive at 42°C, suggesting temperature-dependent activity of FkpA in relation to colicin M processing .

This relationship represents a significant departure from the traditional understanding of periplasmic chaperones, which were previously only implicated in assisting folding and refolding of newly synthesized exported proteins. The requirement of FkpA for colicin M toxicity suggests that this chaperone is involved in the activation of imported proteins, expanding our understanding of its biological roles .

How does temperature affect FkpA function and what are the implications for bacterial physiology?

Temperature significantly impacts FkpA function with important consequences for bacterial physiology:

Experimental evidence reveals that deletion of fkpA causes a 50% reduced biomass yield compared to the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C . Additionally, the ΔfkpA mutants grown at higher temperatures accumulate 7 mM L-glutamate and 22 mM 2-oxoglutarate, indicating significant metabolic disturbances .

The temperature-dependence of FkpA function is further demonstrated in complementation studies with colicin M sensitivity, where temperature-sensitive FkpA mutants show differential activity at different temperatures . The relationship between FkpA and Citrate Synthase (CS) also exhibits temperature dependence, with FkpA having a positive effect on the activity and temperature range of CS in vitro .

These findings suggest that FkpA plays a critical role in bacterial adaptation to temperature stress, likely by maintaining proper protein folding and preventing aggregation of essential proteins at elevated temperatures. This temperature-dependent function may be exploited for improved product formation in biotechnical processes, as suggested by the accumulation of metabolites in ΔfkpA mutants .

What experimental considerations are important when using fkpA Antibody, FITC conjugated for immunofluorescence studies?

When designing immunofluorescence experiments with fkpA Antibody, FITC conjugated, researchers should consider several critical factors:

• Fluorophore Properties and Microscopy Settings:

  • FITC excitation/emission peaks: ~495 nm/~519 nm

  • Use appropriate filter sets to capture FITC signal while avoiding spectral overlap

  • Consider photobleaching effects as FITC is moderately susceptible to photobleaching

• Sample Preparation:

  • Fixation method should preserve antigenic epitopes while maintaining cellular structure

  • Permeabilization is critical for intracellular/periplasmic targets like FkpA

  • Blocking solutions should effectively reduce background without interfering with antibody binding

• Controls:

  • Include proper negative controls (samples lacking the target protein)

  • Use isotype controls (FITC-conjugated rabbit IgG) to assess non-specific binding

  • Consider including FK506 treatment controls to validate specificity (FK506 inhibits FkpA)

• Specificity Validation:

  • Confirm antibody specificity using recombinant FkpA protein as a positive control

  • Validate results with knockout/knockdown models where available

  • Consider cross-reactivity potential, especially when working with bacterial samples containing similar PPIases

• Quantification Considerations:

  • Establish standardized exposure settings for accurate comparison between samples

  • Use appropriate image analysis software for quantifying fluorescence intensity

  • Consider the impact of autofluorescence, particularly from bacterial samples

The antibody has demonstrated high specificity with >95% purity after Protein G purification , but researchers should validate its performance in their specific experimental systems before conducting comprehensive studies.

How can researchers investigate the interaction between FkpA and target proteins using the FITC-conjugated antibody?

Investigating FkpA interactions with target proteins requires methodological approaches that leverage the FITC conjugation. Here are detailed strategies:

• Co-localization Studies:

  • Use the FITC-conjugated fkpA antibody alongside differently-labeled antibodies against potential interacting proteins

  • Employ confocal microscopy to assess spatial overlap with high resolution

  • Analyze co-localization quantitatively using Pearson's correlation coefficient or Manders' overlap coefficient

• Proximity Ligation Assay (PLA):

  • Combine the FITC-conjugated fkpA antibody with primary antibodies against potential interaction partners

  • Use secondary antibodies conjugated with oligonucleotides that can form a circle when in close proximity

  • After ligation and rolling circle amplification, detect fluorescent signals that indicate protein-protein interactions within 40 nm

• Immunoprecipitation Followed by Fluorescence Analysis:

  • Use the antibody for immunoprecipitation of FkpA complexes

  • Leverage the FITC label for direct visualization of precipitated complexes

  • Analyze precipitated proteins using proteomics approaches to identify interaction partners

• FRET-Based Approaches:

  • For advanced applications, combine with CFP-tagged potential interacting proteins

  • FITC can serve as a FRET acceptor for CFP, allowing detection of direct protein interactions

  • Measure FRET efficiency to quantify interaction strength

• In vitro Binding Assays with Purified Components:

  • Use purified FkpA and candidate interacting proteins

  • Monitor interaction by measuring changes in FITC fluorescence upon binding

  • Apply this approach to study the interaction between FkpA and citrate synthase, which has been demonstrated to be functionally relevant

When designing these experiments, researchers should consider the finding that FkpA can delay the aggregation of citrate synthase (CS) and has a positive effect on CS activity and temperature range . This established interaction provides a positive control for developing interaction assays. Additionally, the inhibitory effect of FK506 on FkpA can be used as a control to confirm that observed interactions are specific to FkpA's active form.

What optimization strategies should be employed when using fkpA Antibody, FITC conjugated for western blot analysis?

Optimizing western blot protocols for fkpA Antibody, FITC conjugated requires attention to several technical parameters:

When detecting FkpA from bacterial samples, researchers should consider using specialized lysis buffers that effectively extract periplasmic proteins. The antibody has demonstrated specific detection of recombinant FkpA protein with the expected band size of approximately 55 kDa . For optimal results, researchers should protect samples and membranes from excessive light exposure to prevent FITC photobleaching, and consider using fluorescence-compatible membrane types (such as low-autofluorescence PVDF).

How can researchers validate the specificity of fkpA Antibody, FITC conjugated in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For fkpA Antibody, FITC conjugated, comprehensive validation should include:

• Positive Control Testing:

  • Use purified recombinant FkpA protein as a positive control

  • Confirm single-band detection at the expected molecular weight (approximately 55 kDa)

  • Compare signal intensity across a concentration gradient of recombinant protein

• Competitive Inhibition:

  • Pre-incubate antibody with excess recombinant FkpA

  • Demonstrate signal reduction/elimination in subsequent detection assays

  • Include non-specific protein controls to confirm inhibition specificity

• Genetic Validation:

  • Compare detection between wild-type and fkpA knockout/knockdown models

  • Confirm signal reduction/elimination in knockout samples

  • Rescue experiments with fkpA expression vectors to restore detection

• Pharmacological Validation:

  • Use FK506, which inhibits FkpA activity, to confirm target specificity

  • Determine if FK506 treatment affects antibody binding (it may not if the epitope differs from the FK506 binding site)

  • Compare results with other PPIase inhibitors as controls

• Cross-Reactivity Assessment:

  • Test antibody against related PPIases from the same or different species

  • Evaluate detection in samples from non-target species

  • Confirm specificity for the intended target protein versus related proteins

These validation approaches will ensure that the antibody specifically detects FkpA and not other related proteins or non-specific targets. The high purity (>95%) and affinity purification of the antibody support its specificity , but experimental validation remains essential, particularly when working with complex biological samples or new experimental conditions.

What considerations are important when designing flow cytometry experiments with fkpA Antibody, FITC conjugated?

Flow cytometry with fkpA Antibody, FITC conjugated requires careful experimental design, particularly for bacterial samples:

• Sample Preparation for Bacterial Cells:

  • Fixation protocol optimization to maintain cell integrity while allowing antibody access to periplasmic FkpA

  • Permeabilization methods that preserve bacterial morphology while enabling antibody penetration

  • Single-cell suspension preparation to prevent clumping and ensure accurate measurements

• Fluorescence Parameters:

  • FITC excitation using 488 nm laser

  • Emission collection with 530/30 nm bandpass filter

  • PMT voltage optimization to place negative population appropriately on scale

• Control Samples:

  • Unstained controls to establish autofluorescence baseline

  • FkpA knockout bacteria as negative biological controls

  • Isotype controls (FITC-conjugated rabbit IgG) to assess non-specific binding

  • Single-color controls if performing multicolor experiments

• Compensation and Spectral Considerations:

  • FITC spectral overlap with other fluorophores (particularly PE) requires proper compensation

  • Consider spectral unmixing for complex multicolor panels

  • Account for bacterial autofluorescence in the FITC channel when analyzing data

• Data Analysis:

  • Gating strategy to exclude debris and select intact bacterial cells

  • Consider bacterial cell cycle effects on protein expression levels

  • Population analysis methods to identify FkpA-positive versus negative populations

• Experimental Applications:

  • Quantify FkpA expression under different stress conditions

  • Compare expression levels between wild-type and mutant strains

  • Assess influence of temperature on FkpA expression and localization

When interpreting flow cytometry data, researchers should consider that FkpA is a periplasmic protein in gram-negative bacteria, which requires effective permeabilization for antibody access. Additionally, the relationship between FkpA expression and temperature stress may provide interesting research avenues when analyzing flow cytometry results across different growth conditions.

How can researchers leverage fkpA Antibody, FITC conjugated to study the role of FkpA in bacterial stress responses?

The FITC-conjugated fkpA Antibody offers powerful approaches to investigate FkpA's role in bacterial stress responses:

• Quantitative Stress Response Analysis:

  • Monitor FkpA expression levels under various stress conditions (heat, pH, oxidative stress)

  • Compare expression patterns between wild-type and stress-sensitive mutants

  • Correlate FkpA expression with bacterial survival under stress conditions

• Spatio-temporal Dynamics:

  • Track FkpA localization changes during stress response using time-lapse fluorescence microscopy

  • Analyze potential redistribution of FkpA within the periplasmic space under stress

  • Correlate localization patterns with cellular morphological changes

• Protein-Protein Interaction Networks:

  • Identify stress-dependent FkpA interaction partners using co-immunoprecipitation

  • Map the dynamic interactome of FkpA under normal versus stress conditions

  • Investigate the relationship between FkpA and other chaperones in the stress response network

• Temperature-Dependent Function Studies:

  • Leverage the established temperature-sensitivity of FkpA function

  • Compare protein aggregation patterns between wild-type and ΔfkpA strains at different temperatures

  • Quantify the protective effect of FkpA against heat-induced protein denaturation

• Metabolic Impact Analysis:

  • Investigate how FkpA affects metabolite accumulation under stress conditions

  • Study the relationship between FkpA activity and the accumulation of L-glutamate and 2-oxoglutarate

  • Explore potential biotechnological applications based on metabolic effects

These approaches can provide comprehensive insights into how FkpA contributes to bacterial adaptation to environmental stresses. The finding that FkpA deletion causes a 50% reduced biomass yield at 37°C compared to only 10% at 30°C highlights the protein's critical role in temperature stress adaptation, making this a particularly promising area for research with the FITC-conjugated antibody.

What is the significance of FkpA's effect on citrate synthase activity and how can it be further investigated?

The relationship between FkpA and citrate synthase (CS) represents a significant finding with implications for both basic science and biotechnology:

• Biological Significance:

  • FkpA delays the aggregation of CS, which is inhibited by FK506

  • FkpA positively affects the activity and temperature range of CS in vitro

  • This interaction suggests a direct role for FkpA in maintaining central metabolic enzyme functionality

• Methodological Approaches for Further Investigation:

  • Enzyme Activity Assays:

    • Compare CS activity in the presence and absence of purified FkpA

    • Assess temperature-dependent effects across a range of conditions

    • Determine the stoichiometry of optimal FkpA:CS ratios

  • Structural Analysis:

    • Use FRET-based approaches with labeled FkpA and CS

    • Perform circular dichroism spectroscopy to monitor CS structural changes

    • Apply cryo-EM or X-ray crystallography to the FkpA-CS complex

  • Mutational Analysis:

    • Create targeted mutations in FkpA's chaperone and PPIase domains

    • Assess which domain is responsible for CS interaction and activity enhancement

    • Develop engineered FkpA variants with enhanced CS-stabilizing properties

• Metabolic Consequences:

  • The deletion of fkpA leads to accumulation of 7 mM L-glutamate and 22 mM 2-oxoglutarate

  • This suggests that FkpA impacts TCA cycle flux, potentially through its effect on CS

  • Metabolic flux analysis comparing wild-type and ΔfkpA strains could provide mechanistic insights

• Biotechnological Applications:

  • The FkpA-CS interaction could be exploited for improved product formation in biotechnical processes

  • Enhanced FkpA expression might improve metabolite production in industrial fermentation

  • Co-expression of FkpA with heterologous metabolic pathways could enhance pathway stability

Using the FITC-conjugated fkpA antibody, researchers can track FkpA-CS co-localization in vivo and monitor how this interaction changes under different environmental conditions. This knowledge may lead to novel strategies for metabolic engineering and biotechnological applications.

What are common technical challenges when working with fkpA Antibody, FITC conjugated and how can they be addressed?

Researchers may encounter several technical challenges when working with this antibody:

For optimal results with bacterial samples containing FkpA, researchers should consider:

• For periplasmic protein extraction, use osmotic shock methods rather than whole-cell lysis to enrich for periplasmic proteins

• When analyzing temperature-dependent effects, carefully control experimental temperatures throughout the procedure

• Include FK506 controls to confirm FkpA-specific signals, as FK506 inhibits FkpA activity

• For quantitative comparisons, include recombinant FkpA standards of known concentration

• Account for the potential effects of bacterial growth phase on FkpA expression levels

Addressing these challenges systematically will improve experimental outcomes and data reliability when working with the FITC-conjugated fkpA antibody.

How can researchers differentiate between FkpA's chaperone and PPIase activities in experimental systems?

Differentiating between FkpA's dual functions requires targeted experimental approaches:

• Domain-Specific Inhibition:

  • FK506 specifically inhibits the PPIase activity of FkpA by binding to its C-terminal domain

  • FK506 treatment (12 μM) can be used to selectively inhibit PPIase activity while leaving chaperone function intact

  • Compare experimental outcomes with and without FK506 to identify PPIase-dependent effects

• Domain-Specific Mutations:

  • Design mutations that specifically disrupt either:

    • The α-helical N-domain (chaperone function)

    • The β-pleated sheet C-domain (PPIase activity)

  • Express and purify these mutant proteins for comparative functional assays

  • Use the FITC-conjugated antibody to track localization/expression of these mutants if the epitope remains intact

• Activity-Specific Assays:

  • PPIase Activity: RNase T1 refolding assay (FkpA-His at 35 nM increases refolding 27-fold)

  • Chaperone Activity: Protein aggregation prevention assays (e.g., thermal aggregation of citrate synthase)

  • Compare activity profiles across temperature ranges and with various substrates

• Substrate Specificity Analysis:

  • PPIase activity is specific to proline-containing peptide bonds

  • Test substrates with and without proline residues at key positions

  • Design experiments with proline-rich versus proline-free substrate proteins

• Combined Approaches:

  • Use domain-specific mutants with activity-specific assays

  • Compare wild-type FkpA, domain mutants, and FK506 treatment across multiple experimental readouts

  • Correlate findings with in vivo phenotypes in bacterial systems

These approaches can help researchers attribute specific biological effects to either the chaperone or PPIase activities of FkpA. Understanding this functional separation is particularly important when investigating FkpA's role in stress response, protein folding, and metabolic regulation, as different cellular processes may rely predominantly on one function or the other.

What emerging research areas could benefit from studies using fkpA Antibody, FITC conjugated?

Several cutting-edge research areas could be advanced through studies utilizing this antibody:

• Bacterial Stress Response Networks:

  • Investigate FkpA's role in integrated stress response pathways

  • Map the dynamic interactome of FkpA under various stress conditions

  • Explore potential roles in antibiotic resistance mechanisms

• Synthetic Biology Applications:

  • Engineer enhanced FkpA variants for improved protein folding in biotechnology

  • Develop FkpA-based biosensors for environmental stress detection

  • Utilize FkpA as a stabilizing component in synthetic metabolic pathways

• Host-Pathogen Interactions:

  • Study FkpA's contribution to bacterial virulence and colonization

  • Investigate its role in pathogen survival within host environments

  • Explore potential as a target for anti-virulence therapies

• Metabolic Engineering:

  • Leverage FkpA's effect on citrate synthase for enhanced metabolite production

  • Develop strategies to exploit the glutamate accumulation observed in ΔfkpA strains

  • Design co-expression systems with FkpA to stabilize heterologous metabolic enzymes

• Structural Biology:

  • Elucidate the molecular mechanisms of FkpA's dual functionality

  • Investigate the structural basis of substrate recognition and selectivity

  • Develop structure-based design of FkpA variants with enhanced or altered activity