nfuA Antibody, Biotin conjugated

What is nfuA protein and what is its biological function?

The nfuA protein (also known as Fe/S biogenesis protein NfuA) plays a critical role in iron-sulfur cluster biogenesis. It functions by binding 4Fe-4S clusters and transferring these clusters to apoproteins, thereby participating in the maturation of iron-sulfur (FeS) proteins. Beyond this transfer function, nfuA can also act as a scaffold or chaperone for damaged FeS proteins, providing a protective mechanism within the cell . This dual functionality makes nfuA an important research target for understanding fundamental cellular processes involving iron-sulfur metabolism.

What are the key characteristics of nfuA Antibody, Biotin conjugated?

The nfuA Antibody, Biotin conjugated is a polyclonal antibody produced in rabbit hosts against Escherichia coli Fe/S biogenesis protein NfuA. The specific immunogen used is a recombinant E. coli nfuA protein fragment (amino acids 31-128). The antibody is of IgG isotype and has been purified to >95% purity using Protein G purification methods . The biotin conjugation enables versatile detection methods using streptavidin or avidin systems, similar to other biotin-conjugated antibodies used in research applications .

What is the recommended storage and handling protocol?

For optimal performance and longevity, the nfuA Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity and function . The antibody is supplied in a buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage . When handling biotin-conjugated antibodies, it's advisable to work in reduced light conditions, as extended light exposure can potentially diminish biotin activity.

What applications is nfuA Antibody, Biotin conjugated validated for?

The nfuA Antibody, Biotin conjugated has been specifically validated for ELISA applications . While not explicitly validated for other techniques in the provided data, biotin-conjugated antibodies are generally versatile and can be employed in multiple immunological techniques. Based on similar biotin-conjugated antibodies, potential applications may include immunohistochemistry, immunofluorescence, flow cytometry, and various forms of immunoblotting, provided appropriate protocol optimization and validation .

What controls should be included when using nfuA Antibody, Biotin conjugated?

When designing experiments with nfuA Antibody, Biotin conjugated, several controls are essential:

• Negative controls:

  • Non-immune rabbit IgG (isotype control) at the same concentration as the test antibody

  • Samples known to be negative for nfuA expression

  • Primary antibody omission control to assess secondary reagent specificity

• Positive controls:

  • E. coli lysates (strain K12/DH10B) which express nfuA

  • Recombinant nfuA protein as a reference standard

• Blocking controls:

  • Pre-incubation of the antibody with immunizing peptide to confirm specificity

  • Biotin blocking controls to eliminate potential endogenous biotin interference

These controls help ensure that observed signals are specific and attributable to nfuA protein detection rather than experimental artifacts.

How can researchers optimize signal detection when using biotin-conjugated antibodies?

Optimizing signal detection with biotin-conjugated antibodies requires consideration of several variables:

• Streptavidin/avidin conjugate selection:

  • Choose detection reagents (HRP, fluorophores, gold particles) appropriate for your visualization method

  • Consider using streptavidin instead of avidin for lower background in certain applications

• Signal amplification options:

  • Implement tyramide signal amplification (TSA) for low-abundance targets

  • Use multi-layer detection systems when enhanced sensitivity is required

• Concentration optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Balance signal strength against background levels

Similar to other biotin-conjugated antibodies , researchers should determine the optimal dilution empirically for each specific application and detection system.

How can nfuA Antibody, Biotin conjugated be used to study iron-sulfur cluster biogenesis?

The nfuA Antibody, Biotin conjugated offers several sophisticated approaches for investigating iron-sulfur cluster biogenesis:

• Protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry to identify nfuA binding partners

  • Co-localization studies with other Fe/S cluster assembly components

• Stress response experiments:

  • Monitoring nfuA expression and localization under oxidative stress conditions

  • Comparing wild-type versus mutant nfuA function in cluster transfer

• Comparative analysis across species:

  • Studying conservation of nfuA structure and function across bacterial species

  • Examining evolutionary relationships in Fe/S cluster biogenesis machinery

The antibody can help elucidate how nfuA participates in transferring 4Fe-4S clusters to target apoproteins and how it functions as a scaffold/chaperone for damaged FeS proteins .

What methodological approaches can be used to study nfuA's role as a scaffold/chaperone?

To investigate nfuA's scaffold/chaperone functions for damaged FeS proteins, researchers can employ several methodological approaches:

• In vitro reconstitution assays:

  • Using purified components to reconstruct Fe/S cluster transfer

  • Measuring cluster transfer kinetics with and without nfuA

• Structural biology techniques:

  • Cryo-EM or X-ray crystallography to visualize nfuA-client protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Single-molecule techniques:

  • FRET-based approaches to measure dynamic interactions

  • Optical tweezers to assess mechanical properties of nfuA-client complexes

• Systems biology approaches:

  • Network analysis combining proteomics and transcriptomics data

  • Mathematical modeling of Fe/S cluster assembly pathways

These approaches can provide insights into how nfuA functions as both a transfer protein and a chaperone for damaged FeS proteins .

What are common causes of high background when using nfuA Antibody, Biotin conjugated?

High background is a common challenge when working with biotin-conjugated antibodies. Potential causes and solutions include:

• Endogenous biotin interference:

  • Implement an avidin/biotin blocking step before antibody incubation

  • Use specially formulated blocking reagents designed for biotin-based detection

• Non-specific binding:

  • Optimize blocking conditions (concentration, duration, temperature)

  • Increase washing stringency with appropriate detergents

  • Titrate antibody to find optimal concentration

• Buffer composition issues:

  • Ensure compatibility between the antibody buffer (50% Glycerol, 0.01M PBS, pH 7.4) and experimental conditions

  • Adjust salt concentration to reduce non-specific ionic interactions

• Detection system sensitivity:

  • Reduce exposure/development time to minimize background amplification

  • Select less sensitive detection methods if signal-to-noise ratio is poor

Similar to other biotin-conjugated antibodies, proper optimization of blocking and washing steps is crucial for minimizing background .

How can specificity of nfuA Antibody, Biotin conjugated be validated?

Validating antibody specificity is crucial for experimental rigor. For nfuA Antibody, researchers should consider:

• Molecular validation:

  • Western blot analysis showing single band at expected molecular weight

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Genetic validation:

  • Testing antibody reactivity in nfuA knockout/knockdown models

  • Comparing signal in wild-type versus mutant samples

• Peptide competition:

  • Pre-incubating antibody with the immunizing peptide (nfuA protein fragment 31-128AA)

  • Demonstrating signal reduction with increasing peptide concentration

• Cross-reactivity assessment:

  • Testing against proteins with structural similarity to nfuA

  • Examining reactivity across species boundaries beyond E. coli

These specificity validation steps are particularly important when researching proteins like nfuA that participate in complex interaction networks.

How should quantitative data from nfuA expression studies be normalized?

Proper normalization is essential for meaningful quantitative analysis of nfuA expression:

• Housekeeping protein normalization:

  • Select stable reference proteins appropriate for bacterial systems

  • Use multiple housekeeping proteins for robust normalization

• Total protein normalization:

  • Implement stain-free technology or total protein stains

  • Normalize to total protein rather than single reference proteins

• Sample-specific considerations:

  • Account for growth phase effects on reference gene expression

  • Consider normalization to cell number for unicellular organisms

• Statistical approaches:

  • Apply geometric averaging of multiple reference genes

  • Use algorithmic approaches to identify most stable normalizers

What considerations are important when comparing nfuA expression levels across different experimental conditions?

When analyzing nfuA expression across different experimental conditions, researchers should consider:

• Iron availability effects:

  • Monitor and report iron concentrations in growth media

  • Establish standardized iron supplementation protocols

• Oxidative stress interactions:

  • Measure and account for oxidative stress markers

  • Consider the interplay between redox state and Fe/S protein stability

• Growth phase dependencies:

  • Standardize sampling points based on growth curve rather than absolute time

  • Account for growth phase-dependent regulation of Fe/S metabolism

• Technical variation sources:

  • Implement technical replicates to assess method reproducibility

  • Account for batch effects in multi-day experiments

• Biological interpretation frameworks:

  • Integrate nfuA expression data with functional assays of Fe/S protein activity

  • Consider pathway-level analysis rather than focusing solely on nfuA

These considerations help ensure that observed differences in nfuA expression are biologically meaningful rather than artifacts of experimental variation.