grxD Antibody, Biotin conjugated

What is grxD protein and what biological functions does it serve in bacterial systems?

Glutaredoxin 4 (grxD/Grx4) is a monothiol glutaredoxin containing a conserved CGFS motif that plays essential roles in bacterial physiology. It functions primarily in:

• Iron-sulfur ([Fe-S]) cluster biogenesis and maturation

• Protection against oxidative stress

• Iron homeostasis regulation

• Virulence factor regulation in pathogenic bacteria

In Escherichia coli K12 strain, grxD (UniProt ID: P0AC69) consists of 115 amino acids and functions as a thiol-disulfide oxidoreductase that maintains cellular redox balance . Studies in Pseudomonas aeruginosa have demonstrated that grxD performs crucial roles in oxidative stress protection and acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during cumene hydroperoxide degradation .

What are the key specifications of biotin-conjugated grxD antibody?

The biotin-conjugated grxD antibody represents a specialized research tool with the following specifications:

The biotin conjugation provides significant advantages for detection sensitivity and versatility in immunoassays compared to non-conjugated versions .

How does the storage and handling of biotin-conjugated grxD antibody differ from standard antibodies?

Proper storage of biotin-conjugated grxD antibody requires special considerations:

• Temperature requirements: Store at -20°C or -80°C immediately upon receipt .

• Freeze-thaw minimization: Aliquot the antibody upon first thaw to minimize repeated freeze-thaw cycles that can degrade the biotin conjugate and reduce sensitivity .

• Buffer composition stabilization: The standard buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) is specifically formulated to maintain both antibody integrity and biotin conjugation stability .

• Light protection: Store in amber tubes or wrapped in aluminum foil to protect the biotin conjugate from light exposure, which can reduce signal intensity over time.

• Working dilution preparation: When preparing working dilutions, use freshly prepared buffers free of contaminating biotin sources that could compete for streptavidin binding sites during detection.

What are the optimal protocols for using biotin-conjugated grxD antibody in ELISA applications?

When employing biotin-conjugated grxD antibody in ELISA applications, researchers should follow these methodological considerations:

• Plate preparation: Use high-binding microplates coated with capture antigen or sample at 1-10 μg/mL in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking optimization: Block with 1-3% BSA in PBS for 1-2 hours at room temperature to minimize non-specific binding.

• Antibody dilution determination:

  • Start with a titration series from 1:1000 to 1:5000 dilutions to determine optimal signal-to-noise ratio .

  • For biotinylated versions specifically, begin with more dilute concentrations (1:2000-1:5000) since detection amplification through streptavidin systems compensates for lower antibody concentrations.

• Detection system selection:

  • Utilize streptavidin-HRP (recommended dilution 1:10,000 to 1:20,000)

  • Alternative detection systems include streptavidin-alkaline phosphatase or streptavidin-fluorophore conjugates for specialized applications

• Signal optimization:

  • Incubation time: 1-2 hours at room temperature or overnight at 4°C

  • Washing steps: Minimum 4-5 washes with PBS containing 0.05% Tween-20

• Positive control inclusion: Include purified recombinant grxD protein as positive control to validate assay performance.

How can researchers validate the specificity of grxD antibodies in bacterial samples?

Comprehensive validation of antibody specificity requires multiple approaches:

• Western blot analysis:

  • Compare wild-type bacterial lysate against a ΔgrxD mutant strain

  • Expected molecular weight for E. coli grxD: approximately 12.2 kDa

  • Recommended dilution range: 1:1000-1:5000

• Positive control verification:

  • Use purified recombinant grxD protein (1-115AA or 24-115AA fragment) as positive control

  • Perform peptide competition assay where pre-incubation with immunizing peptide should abolish signal

• Cross-reactivity assessment:

  • Test against closely related bacterial species to confirm species specificity

  • Evaluate against other glutaredoxin family members (grxA, grxB, grxC) to confirm isoform specificity

• Gene knockout validation:

  • The definitive control involves comparing signal between wild-type and genetically confirmed grxD deletion mutants

  • Complementation strains (ΔgrxD/pGrxD) should restore signal to wild-type levels

• Mass spectrometry verification:

  • For advanced validation, perform immunoprecipitation followed by mass spectrometry analysis to confirm target identity

How does the CGFS motif in grxD contribute to its function in iron-sulfur cluster biogenesis?

The conserved CGFS motif (specifically C29GFS in P. aeruginosa) represents the catalytic center of grxD and plays several critical roles:

• Mechanistic contributions:

  • The cysteine residue (C29) is essential for coordinating iron in [Fe-S] clusters as demonstrated through site-directed mutagenesis studies

  • Mutation of this cysteine abolishes the protein's ability to regulate downstream processes including 2,4-DAPG production in P. fluorescens

• Functional evidence:

  • In P. aeruginosa, site-directed mutagenesis of the CGFS motif eliminated grxD's ability to protect against oxidative stress

  • The cysteine residue specifically contributes to the protein's function in [Fe-S] cluster maturation pathways

• Structural implications:

  • Unlike dithiol glutaredoxins, monothiol glutaredoxins with the CGFS motif cannot efficiently catalyze classical glutaredoxin reactions

  • Instead, the CGFS motif specializes the protein for iron metabolism functions and [Fe-S] cluster trafficking

• Experimental approaches to study the motif:

  • Site-directed mutagenesis replacing cysteine with serine (CGFS→SGFS) provides a catalytically inactive control

  • Complementation studies with mutated versions can determine which functions require the intact CGFS motif

What is the relationship between grxD expression and oxidative stress response in bacteria?

The relationship between grxD and oxidative stress is multifaceted and can be experimentally investigated through several approaches:

• Phenotypic characterization:

  • ΔgrxD mutants show hypersensitivity to oxidative stress-generating agents including paraquat (PQ) and cumene hydroperoxide (CHP)

  • Growth curve analysis reveals that ΔgrxD mutants grow more slowly during log phase compared to wild-type strains, with complementation strains restoring normal growth

• Molecular mechanism:

  • grxD acts as an electron donor for the organic hydroperoxide resistance enzyme (Ohr) during hydroperoxide degradation

  • The protein functions as an [Fe-S] cluster carrier involved in [Fe-S] cluster maturation, protecting these sensitive clusters from oxidative damage

• Transcriptional regulation:

  • ΔgrxD mutants show constitutively high expression of oxidative stress-responsive genes including iscR (2.4-fold), nfuA (2.1-fold), tpx (2.1-fold), and fprB (2.8-fold)

  • This suggests a compensatory mechanism where the absence of grxD triggers upregulation of other oxidative stress protection systems

• Experimental approaches:

  • Challenge bacteria with graduated concentrations of oxidants (H₂O₂, paraquat, cumene hydroperoxide)

  • Measure survival rates, growth inhibition zones, and enzymatic activity

  • Monitor expression of oxidative stress genes using qRT-PCR or reporter constructs

How does deletion of grxD affect virulence and pathogenicity in bacterial systems?

The impact of grxD on bacterial virulence has been demonstrated in multiple pathogenic species:

• Pseudomonas aeruginosa virulence:

  • ΔgrxD mutants demonstrate attenuated virulence in a Drosophila melanogaster host model

  • The reduced virulence correlates with increased sensitivity to oxidative stress, which is a key host defense mechanism

• Pathogenicity mechanisms:

  • In Pseudomonas fluorescens, grxD regulates the production of 2,4-diacetylphloroglucinol (2,4-DAPG), an important antifungal compound

  • The protein influences exoprotease production through the RsmA and RsmE regulatory proteins

  • Deletion of grxD increases expression of phlF, which negatively regulates 2,4-DAPG production

• Biocontrol applications:

  • The grxD mutant shows severely impaired biocontrol effect against bacterial wilt of tomato, demonstrating its importance for beneficial plant-microbe interactions

• Experimental approaches:

  • In vitro virulence assays: Measure production of virulence factors like exoproteases, siderophores, and toxins

  • Cell culture models: Assess bacterial adhesion, invasion, and cytotoxicity in relevant host cell lines

  • Animal infection models: Compare wild-type and ΔgrxD mutant strains in appropriate infection models

  • Transcriptome analysis: Identify virulence-associated genes regulated by grxD

What methodological approaches can resolve contradictory results when using grxD antibody in different bacterial species?

When encountering contradictory results across bacterial species, researchers should implement systematic troubleshooting:

• Epitope conservation analysis:

  • Perform sequence alignment of grxD proteins across studied bacterial species

  • Identify variations in the antibody's target epitope region that might affect binding affinity

  • Consider generating species-specific antibodies if epitope conservation is low

• Expression level normalization:

  • Quantify grxD expression levels in different species using qRT-PCR

  • Adjust antibody concentrations or detection sensitivity accordingly

  • Use housekeeping protein controls specific to each bacterial species

• Protocol optimization matrix:

  • Systematically test multiple variables in parallel:

    • Lysis buffer composition (detergent type/concentration, salt concentration)

    • Antibody dilution series (1:500 to 1:5000)

    • Incubation conditions (temperature, duration)

    • Detection system sensitivity

• Cross-validation approaches:

  • Employ alternative detection methods (mass spectrometry, activity assays)

  • Generate knockout controls in each bacterial species

  • Utilize epitope-tagged recombinant proteins for calibration curves

• Data integration framework:

  • Document all experimental conditions systematically

  • Use statistical methods to normalize results across species

  • Consider developing a correction factor based on sequence homology

How can biotin-conjugated grxD antibody be utilized in advanced protein-protein interaction studies?

Biotin-conjugated grxD antibody enables sophisticated protein interaction studies:

• Pull-down assay optimization:

  • Use streptavidin-coated magnetic beads to capture biotin-conjugated grxD antibody

  • Pre-form antibody-antigen complexes before adding to lysate to minimize non-specific binding

  • Include appropriate controls: non-biotinylated antibody, irrelevant biotinylated antibody, and pre-clearing steps

• Co-immunoprecipitation (Co-IP) protocols:

  • Optimize cell lysis conditions to preserve native protein complexes

  • Consider mild detergents (0.1% NP-40 or digitonin) to maintain interactions

  • Validate interaction partners through reciprocal Co-IP and mass spectrometry

• Proximity-dependent labeling approaches:

  • Combine with BioID or APEX2 proximity labeling techniques

  • Design fusion proteins with grxD to identify proximal interacting partners

  • Analyze labeled proteins using streptavidin pulldown followed by mass spectrometry

• Microscopy applications:

  • Use biotin-conjugated grxD antibody with fluorescent streptavidin conjugates

  • Implement super-resolution microscopy to visualize grxD co-localization with potential partner proteins

  • Perform Förster Resonance Energy Transfer (FRET) analysis using appropriate fluorophore pairs

• Quantitative interaction analysis:

  • Develop Surface Plasmon Resonance (SPR) protocols using biotin-conjugated antibody

  • Measure binding kinetics and affinity constants between grxD and proposed interaction partners

  • Perform competition assays to identify binding interfaces

What techniques can be used to study the interaction between grxD and iron-sulfur cluster assembly machinery?

Studying grxD's role in iron-sulfur cluster assembly requires specialized approaches:

• Reconstitution assays:

  • Purify recombinant grxD protein and iron-sulfur cluster assembly components

  • Monitor [Fe-S] cluster formation spectroscopically (UV-visible absorption at ~420 nm)

  • Track iron incorporation using radioactive iron (⁵⁵Fe) or colorimetric assays

• Genetic interaction mapping:

  • Create double mutants between grxD and other [Fe-S] carrier proteins (ErpA, IscA, NfuA)

  • Analyze synthetic phenotypes under oxidative stress conditions

  • Test complementation with ectopic expression of [Fe-S] carrier proteins in ΔgrxD mutants

• Protein complex isolation:

  • Employ tandem affinity purification with tagged grxD

  • Use chemical crosslinking to stabilize transient interactions

  • Analyze complexes by blue native PAGE followed by mass spectrometry

• Activity assays for [Fe-S] dependent enzymes:

  • Measure activities of model [Fe-S] enzymes (aconitase, fumarase) in wild-type vs. ΔgrxD strains

  • Monitor cluster transfer from grxD to recipient proteins in vitro

  • Develop high-throughput screening assays for [Fe-S] cluster assembly

• Advanced imaging techniques:

  • Use transmission electron microscopy with immunogold labeling

  • Implement correlative light and electron microscopy (CLEM)

  • Develop FRET-based sensors for monitoring [Fe-S] cluster transfer events