CpNIFS3 Antibody

What is CpNIFS3 and why is it important in plant research?

CpNIFS3 (Chloroplast NIFS-like 3) is a protein encoded by the Arabidopsis thaliana genome with UniProt accession number Q3E6S9 . This protein belongs to the NifS-like family of proteins, which are critical for iron-sulfur cluster assembly in chloroplasts. Iron-sulfur clusters are essential cofactors for many enzymes involved in photosynthesis, respiration, and nitrogen fixation in plants.

The study of CpNIFS3 is important for understanding chloroplast biogenesis, photosynthetic efficiency, and plant responses to environmental stresses. Antibodies against CpNIFS3 are valuable tools for investigating protein localization, expression levels, and post-translational modifications in different plant tissues and under various experimental conditions.

What are the key applications for CpNIFS3 antibodies in plant research?

CpNIFS3 antibodies can be applied in several fundamental research techniques:

• Western blotting for protein detection and quantification

• Immunoprecipitation for protein-protein interaction studies

• Immunohistochemistry and immunofluorescence for localization studies

• ChIP assays if the protein has DNA-binding capabilities

• ELISA for quantitative protein measurements

These applications enable researchers to investigate CpNIFS3's role in chloroplast development, its interactions with other proteins involved in iron-sulfur cluster assembly, and its expression patterns under different environmental conditions or in various mutant backgrounds.

How should CpNIFS3 antibody be stored and handled to maintain reactivity?

For optimal performance, CpNIFS3 antibodies should be stored according to manufacturer recommendations, typically at -20°C for long-term storage with minimal freeze-thaw cycles . For working solutions, storage at 4°C for up to one month is generally acceptable.

When handling the antibody:

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Use sterile techniques when handling to prevent contamination

• Allow the antibody to reach room temperature before opening the vial

• Gently mix by inversion rather than vortexing to prevent protein denaturation

• Ensure proper labeling with reception date and opening date

• Consider adding preservatives like sodium azide (0.02%) for longer storage at 4°C

Proper storage and handling are essential for maintaining antibody specificity and sensitivity in experimental applications.

How should positive and negative controls be selected for CpNIFS3 antibody validation?

Proper control selection is critical for validating CpNIFS3 antibody specificity and performance. For positive controls, consider:

• Recombinant CpNIFS3 protein for direct verification

• Arabidopsis thaliana wild-type leaf extracts where CpNIFS3 is known to be expressed

• Transgenic lines overexpressing CpNIFS3 with an epitope tag

For negative controls, researchers should implement:

• CpNIFS3 knockout or knockdown lines (T-DNA insertion lines or CRISPR-edited plants)

• Non-plant tissues where the target protein is absent

• Pre-immune serum for polyclonal antibodies

• Isotype control for monoclonal antibodies

• Peptide competition assays where the antibody is pre-incubated with the immunizing peptide

Proper control selection helps distinguish specific signals from background and non-specific interactions, which is particularly important given the potential for cross-reactivity with other NifS family members in Arabidopsis .

What are the optimal protein extraction protocols for detecting CpNIFS3 in plant tissues?

Given CpNIFS3's localization in chloroplasts, extraction protocols should preserve chloroplast proteins while minimizing interference from abundant photosynthetic proteins. A recommended protocol includes:

• Harvest fresh plant tissue and immediately freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining frozen state

• Extract in buffer containing:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 10% glycerol

  • 1% Triton X-100 or NP-40

  • 1 mM EDTA

  • 1 mM PMSF and protease inhibitor cocktail

  • 5 mM DTT or β-mercaptoethanol

• Centrifuge at 12,000 g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

• Add SDS sample buffer and heat at 95°C for 5 minutes

This extraction method helps maintain protein integrity while solubilizing membrane-associated proteins. For subcellular fractionation to isolate chloroplasts before extraction, additional steps would be required to ensure enrichment of the target compartment.

What is the recommended dilution range for CpNIFS3 antibody in different applications?

Optimal antibody dilution must be determined empirically for each application and experimental system. Based on standard practices for similar plant antibodies, these starting ranges are recommended:

It's advisable to perform a titration experiment to determine the optimal antibody concentration that provides the best signal-to-noise ratio for your specific experimental conditions. The manufacturer's recommended dilution (if provided) should be used as a starting point .

How can specificity of CpNIFS3 antibody be verified when working with closely related NifS family proteins?

Verifying specificity is particularly challenging with protein families that share high sequence homology. For CpNIFS3 antibody, these advanced approaches can help establish specificity:

• Epitope mapping to identify the exact binding region of the antibody

• Cross-reactivity testing against recombinant versions of related NifS family proteins

• Mass spectrometry analysis of immunoprecipitated proteins to confirm identity

• Comparative analysis using multiple antibodies raised against different epitopes of CpNIFS3

• Testing in genetic backgrounds with selective knockouts of related family members

The biophysics-informed modeling approach described in recent literature can be particularly valuable for disentangling binding specificity patterns in antibodies that might recognize closely related epitopes . This approach associates each potential ligand with a distinct binding mode, enabling prediction of cross-reactivity and specificity profiles.

What approaches can resolve contradictory results when using CpNIFS3 antibody across different experimental conditions?

When facing contradictory results, systematic troubleshooting is essential:

• Verify antibody performance with fresh aliquots and positive controls

• Assess protein extraction efficiency across different tissues or conditions

• Consider post-translational modifications that might affect epitope accessibility

• Evaluate buffer conditions that might impact antibody binding

• Implement alternative detection methods to corroborate findings

• Use orthogonal approaches (e.g., fluorescent protein tagging, mass spectrometry)

• Examine technical variables (incubation times, temperatures, blocking agents)

If specific contradictions emerge related to antibody binding characteristics, consider that different monoclonal antibodies against the same protein complex can display markedly different functional effects despite binding to the same receptor, as demonstrated in neutrophil studies . This principle may apply to plant protein complexes as well.

How can CpNIFS3 antibody be utilized in co-immunoprecipitation studies to identify interaction partners?

For effective co-immunoprecipitation (Co-IP) studies with CpNIFS3 antibody:

• Optimize protein extraction conditions to preserve protein-protein interactions:

  • Use gentle detergents (0.5-1% NP-40 or Digitonin)

  • Include stabilizing agents (glycerol, EDTA)

  • Maintain physiological pH (7.0-7.5)

  • Consider crosslinking to stabilize transient interactions

• Technical approach:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate cleared lysates with CpNIFS3 antibody (2-5 μg per mg of protein)

  • Add protein A/G beads and incubate with gentle rotation

  • Wash extensively with decreasing detergent concentrations

  • Elute protein complexes and analyze by mass spectrometry or Western blotting

• Controls to include:

  • Input samples to verify starting material

  • IgG control to identify non-specific interactions

  • Reverse Co-IP with antibodies against suspected partners

  • Verification in knockout/knockdown lines

This methodology can identify novel protein interactions in iron-sulfur cluster assembly pathways, potentially revealing new components of chloroplast metabolic networks.

What are common sources of background signal when using CpNIFS3 antibody and how can they be minimized?

Plant tissues present unique challenges for antibody-based detection due to abundant photosynthetic pigments, polyphenols, and other interfering compounds. Common sources of background and their solutions include:

• Non-specific antibody binding:

  • Optimize blocking conditions (5% BSA or milk in TBST)

  • Include 0.1-0.5% Tween-20 in wash buffers

  • Pre-adsorb antibody with plant extracts from negative control tissue

• Endogenous peroxidase activity:

  • Include quenching steps (3% H₂O₂ treatment) before antibody incubation

  • Use alternative detection methods like fluorescence

• Autofluorescence from chlorophyll and other plant pigments:

  • Use appropriate spectral filters

  • Consider counterstaining techniques

  • Implement spectral unmixing in microscopy

• Cross-reactivity with related proteins:

  • Use peptide competition assays

  • Optimize antibody dilution

  • Consider monoclonal antibodies for higher specificity

Careful optimization of these parameters can significantly improve signal-to-noise ratio when working with CpNIFS3 antibody in plant tissues.

How should quantitative data from CpNIFS3 immunoblots be normalized for accurate comparison across samples?

Reliable quantification requires careful normalization strategies:

• Loading control selection:

  • Housekeeping proteins (tubulin, actin) may not be ideal for all conditions

  • Consider multiple loading controls

  • Evaluate total protein staining methods (Ponceau S, SYPRO Ruby)

• Technical considerations:

  • Use linear range of detection for both target and reference proteins

  • Implement technical replicates

  • Validate antibody linearity over the concentration range of interest

• Normalization method:

  • Calculate relative density ratios (CpNIFS3/loading control)

  • Consider normalization to total protein when appropriate

  • For tissue-specific or developmental studies, identify tissue-specific references

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological replicates (n≥3) for meaningful comparisons

  • Report both raw and normalized values for transparency

This methodological approach ensures that observed differences in CpNIFS3 levels represent biological variation rather than technical artifacts.

What methodological approaches can distinguish between post-translational modifications of CpNIFS3 protein?

Post-translational modifications (PTMs) of CpNIFS3 may significantly impact its function in iron-sulfur cluster assembly. To investigate these modifications:

• Specialized extraction techniques:

  • Include phosphatase inhibitors for phosphorylation studies

  • Add deacetylase inhibitors for acetylation studies

  • Use reducing/non-reducing conditions to examine disulfide bonding

• Analytical approaches:

  • 2D gel electrophoresis to separate protein isoforms

  • Phos-tag gels for mobility shift of phosphorylated proteins

  • Western blotting with modification-specific antibodies (anti-phospho, anti-acetyl)

  • Mass spectrometry for comprehensive PTM mapping

• Functional validation:

  • Site-directed mutagenesis of modified residues

  • In vitro enzymatic assays with and without modifications

  • Correlation of modification status with functional outcomes

These approaches can reveal how PTMs regulate CpNIFS3 activity under different physiological conditions or developmental stages, providing insights into the regulation of iron-sulfur cluster assembly in plants.

How can CpNIFS3 antibody studies be complemented with genetic approaches for comprehensive functional analysis?

Integrating immunological and genetic approaches provides powerful insights into CpNIFS3 function:

• Genetic resources to combine with antibody studies:

  • T-DNA insertion mutants of CpNIFS3 and related genes

  • CRISPR/Cas9-generated knockout or knockdown lines

  • Conditional expression systems (inducible promoters)

  • Complementation lines with tagged versions of CpNIFS3

• Integrated experimental approaches:

  • Protein expression/localization studies in genetic backgrounds

  • Phenotypic characterization correlated with protein levels

  • Epistasis analysis combined with protein interaction studies

  • Stress response experiments with protein-level monitoring

• Synthetic biology approaches:

  • Domain swapping between related NifS proteins

  • Structure-function studies guided by antibody epitope mapping

  • Orthogonal labeling systems combined with immunodetection

This integration allows researchers to connect molecular-level observations with physiological outcomes, strengthening causal relationships in CpNIFS3 functional studies.

What considerations apply when using CpNIFS3 antibody across different plant species or heterologous expression systems?

Cross-species applications require careful validation:

• Sequence conservation analysis:

  • Align CpNIFS3 sequences across species of interest

  • Identify conservation at the epitope region (if known)

  • Predict potential cross-reactivity based on homology

• Validation strategies:

  • Western blot against recombinant proteins from each species

  • Peptide competition assays with species-specific peptides

  • Graduated testing from closely to distantly related species

• Heterologous expression considerations:

  • Codon optimization for expression host

  • Subcellular targeting in non-plant systems

  • Post-translational modification differences

  • Protein folding variations affecting epitope accessibility

When using the antibody in heterologous systems, empirical validation is essential, as even highly conserved proteins may show different antibody reactivity due to subtle differences in protein structure or post-translational modifications .

How can researchers contribute to improving CpNIFS3 antibody resources for the scientific community?

Improving antibody resources requires collective effort:

• Characterization and validation:

  • Document antibody performance in diverse applications

  • Define epitope regions through mapping studies

  • Establish detection limits and linear ranges

• Data sharing practices:

  • Publish detailed methods including antibody dilutions, incubation times, and buffer compositions

  • Deposit validation data in public repositories

  • Include comprehensive controls in publications

• Community resources:

  • Contribute to antibody validation databases

  • Participate in multi-laboratory validation studies

  • Share protocols through community forums