ABCI7 Antibody

What is ABCI7 and what is its molecular function in plant systems?

ABCI7, also known as ATP-binding cassette I7, ATNAP6, NAP6, or non-intrinsic ABC protein 6, is a member of the ABC transporter family in plants, particularly characterized in Arabidopsis thaliana. The protein is encoded by gene loci F5D14.28/F5D14_28 . Unlike mammalian ABCB7 (which exports iron-sulfur clusters from mitochondria to cytosol ), plant ABCI7 has distinct functions in plant cellular processes.

As an ABC transporter component, ABCI7 likely participates in energy-dependent transport of substrates across membranes, utilizing ATP hydrolysis. Research suggests its involvement in metal ion homeostasis and potential roles in stress response pathways. The protein's structure includes characteristic ATP-binding domains that enable its transport functions.

Methodological approach: To properly characterize ABCI7 function, researchers should employ multiple complementary techniques including gene expression analysis (qRT-PCR), protein localization studies (using fluorescent protein fusions), and phenotypic analysis of knockout/knockdown mutants.

What experimental applications is the ABCI7 antibody validated for?

The ABCI7 antibody from CUSABIO Technology LLC (catalog number CSB-PA878548XA01DOA) has been validated for several research applications:

When designing experiments, researchers should note that this antibody has been specifically validated with recombinant protein/peptide immunogen rather than endogenous protein in complex biological samples .

What are the optimal storage and handling conditions for maintaining ABCI7 antibody integrity?

The ABCI7 antibody requires specific storage conditions to maintain its functionality and specificity. Upon receipt, the antibody should be stored at either -20°C or -80°C, with -80°C being preferable for long-term storage . Repeated freeze-thaw cycles should be strictly avoided as they can lead to antibody degradation and loss of binding efficiency.

The antibody is formulated with preservative (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) in liquid format . When working with this antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Thaw aliquots at 4°C rather than room temperature

• Keep on ice during experiment preparation

• Return to -20°C or -80°C immediately after use

• Avoid exposure to light for extended periods

For experimental use, it is recommended to dilute working solutions in fresh buffer containing 1% BSA as a blocking agent to minimize non-specific binding.

What controls should be incorporated when using ABCI7 antibody in immunological experiments?

Proper experimental controls are essential for reliable interpretation of results when using ABCI7 antibody:

• Positive control: The antibody kit includes 200μg recombinant immunogen protein/peptide that should be used as a positive control to verify antibody performance .

• Negative control: The pre-immune serum (1ml) provided with the antibody should be utilized as a negative control to establish baseline and non-specific binding levels .

• Isotype control: A non-specific rabbit IgG at the same concentration should be used to identify potential background resulting from the antibody isotype.

• Genetic controls: When possible, include samples from ABCI7 knockout or knockdown plants to confirm antibody specificity.

• Blocking peptide control: A competitive binding assay using excess immunizing peptide can verify signal specificity.

The inclusion of these controls enables proper validation of experimental results and supports reproducibility in scientific research.

How can ABCI7 antibody be optimized for immunolocalization in plant tissues?

Immunolocalization of ABCI7 in plant tissues presents unique challenges due to plant cell wall barriers and potential epitope masking. To optimize this application:

• Fixation protocol optimization: Compare multiple fixation protocols including:

  • Paraformaldehyde (3-4%) fixation for 2-4 hours

  • Ethanol:acetic acid (3:1) for improved penetration

  • Methanol fixation for certain membrane proteins

• Cell wall digestion: Implement a balanced enzymatic digestion using:

  • Cellulase (1-2%) and Macerozyme (0.2-0.5%) cocktail

  • Pectinase treatment (0.1-0.3%)

  • Monitor digestion carefully to prevent overdigestion and tissue damage

• Antibody penetration enhancement:

  • Use extended incubation periods (12-24 hours at 4°C)

  • Include 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 in blocking buffer

  • Consider vacuum infiltration of antibody solutions

• Signal amplification:

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

  • Use highly cross-adsorbed secondary antibodies to minimize background

  • Optimize antibody concentration through systematic dilution series (1:100 to 1:2000)

Validation through parallel approaches such as ABCI7-fluorescent protein fusions can help confirm localization patterns observed with antibody-based detection.

What are the methodological considerations for using ABCI7 antibody in co-immunoprecipitation (Co-IP) studies?

Co-immunoprecipitation with ABCI7 antibody can identify protein interaction partners, providing insight into functional networks. Key methodological considerations include:

• Extraction buffer optimization:

  • Test multiple buffer compositions (HEPES, Tris, phosphate-based)

  • Adjust salt concentration (100-500mM) to balance specificity and yield

  • Include appropriate detergents (0.5-1% NP-40, 0.1-0.5% Triton X-100)

  • Add protease inhibitor cocktail and phosphatase inhibitors if studying phosphorylation states

• Crosslinking considerations:

  • For transient interactions, implement mild crosslinking (0.1-0.5% formaldehyde)

  • Use membrane-permeable crosslinkers like DSP for organelle-localized interactions

  • Include crosslink reversal controls

• Antibody coupling strategies:

  • Direct coupling to protein A/G beads may improve yield

  • Test both pre-clearing sample with beads and pre-binding antibody to beads

  • Consider orientation-specific coupling to avoid blocking antigen recognition sites

• Validation approaches:

  • Perform reverse Co-IP with antibodies against suspected interaction partners

  • Include IgG controls and ABCI7-knockout/knockdown samples

  • Confirm interactions using orthogonal methods (Y2H, BiFC, FRET)

How can researchers address epitope accessibility issues when using ABCI7 antibody in different subcellular fractions?

Epitope accessibility is a critical factor affecting ABCI7 antibody performance across different subcellular fractions. Since the antibody was generated against a recombinant protein , its performance may vary depending on protein conformation and environmental context.

• Epitope mapping considerations:

  • The antibody was generated against the full ABCI7 recombinant protein

  • Predict potentially hidden epitopes using protein structure prediction tools

  • Consider different extraction methods that may expose different epitopes

• Subcellular fraction-specific protocols:

  • For membrane fractions: Include 0.5-1% SDS or 8M urea to denature proteins

  • For nuclear fractions: Use sonication and nuclease treatment

  • For chloroplast/mitochondrial fractions: Implement osmotic shock methods

• Denaturation state optimization:

  • For Western blot: Compare reducing vs. non-reducing conditions

  • Test different heating protocols (65°C vs. 95°C; 5 min vs. 10 min)

  • Evaluate the effects of different detergents (SDS, Triton X-100, CHAPS)

• Experimental validation approach:

  • Compare native vs. denatured protein detection efficiency

  • Generate subcellular markers as parallel controls

  • Verify localization using orthogonal methods (fluorescent protein fusions)

By systematically investigating these parameters, researchers can optimize detection of ABCI7 across different subcellular compartments and experimental conditions.

What quantitative approaches can be used to analyze ABCI7 expression in response to environmental stressors?

Quantitative analysis of ABCI7 expression under stress conditions requires robust methodological approaches:

• Western blot quantification protocol:

  • Implement standardized loading controls (anti-actin, anti-tubulin)

  • Use infrared fluorescent secondary antibodies for wider linear range

  • Perform technical triplicates with biological replicates (n≥3)

  • Analyze using densitometry software with background subtraction

  • Present data as fold-change relative to control conditions

• ELISA-based quantification:

  • Develop a standard curve using recombinant ABCI7 protein (purified)

  • Implement sandwich ELISA for increased sensitivity

  • Normalize protein loading across samples before analysis

  • Calculate absolute protein quantities based on standard curve

• Stress response experimental design:

  • Time-course analysis (0, 1, 3, 6, 12, 24, 48 hours post-treatment)

  • Dose-response experiments with multiple stress intensities

  • Combine different stressors to identify interaction effects

  • Include recovery phases to assess protein turnover

• Statistical analysis approach:

  • Apply appropriate statistical tests (ANOVA, t-test)

  • Implement multiple testing correction for time-course/dose-response

  • Calculate effect sizes and confidence intervals

  • Determine biological significance thresholds (typically >1.5-fold change)

Example data representation format:

How can ABCI7 antibody be used to investigate protein-protein interactions within the ABC transporter complex?

Investigating ABCI7's interactions within larger ABC transporter complexes requires sophisticated approaches:

• Proximity-based protein interaction analysis:

  • Implement BioID or TurboID proximity labeling using ABCI7 as bait

  • Employ ABCI7 antibody for pulldown of biotinylated interaction partners

  • Use chemical crosslinking followed by immunoprecipitation with ABCI7 antibody

  • Validate with reciprocal pulldowns using antibodies against identified partners

• Native complex isolation techniques:

  • Blue Native PAGE followed by Western blotting with ABCI7 antibody

  • Sucrose gradient fractionation to separate intact complexes

  • Size-exclusion chromatography coupled with immunodetection

  • Glycerol gradient ultracentrifugation for complex separation

• Interaction domain mapping:

  • Generate domain-specific constructs for deletion analysis

  • Use peptide competition assays to identify binding interfaces

  • Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Conduct alanine scanning mutagenesis of potential interaction domains

• Functional validation of interactions:

  • Measure transport activity in reconstituted liposomes

  • Assess ATP hydrolysis rates of purified complexes

  • Analyze phenotypic changes in plants with disrupted interaction sites

  • Monitor subcellular localization changes upon interaction disruption

These approaches can be integrated into a multifaceted experimental pipeline to characterize the composition, stoichiometry, and functional significance of ABCI7-containing complexes.

How can researchers address non-specific binding issues when using ABCI7 antibody in Western blot applications?

Non-specific binding is a common challenge when working with antibodies in complex plant extracts. To address this issue with ABCI7 antibody:

• Blocking optimization:

  • Compare different blocking agents (5% BSA, 5% non-fat milk, commercial blockers)

  • Extend blocking time (2-16 hours at 4°C)

  • Test different blocking buffer compositions (TBS vs. PBS base)

  • Include 0.1-0.3% Tween-20 in wash and antibody incubation buffers

• Antibody dilution optimization:

  • Perform systematic dilution series (1:500 to 1:5000)

  • Test extended primary antibody incubation at 4°C (overnight to 48 hours)

  • Reduce secondary antibody concentration if background persists

  • Implement thorough washing steps (5-6 washes of 10 minutes each)

• Sample preparation refinement:

  • Include additional purification steps (ion exchange, size exclusion)

  • Pre-clear lysates with Protein A/G beads to remove sticky proteins

  • Treat samples with nucleases to reduce nucleic acid interference

  • Optimize protein extraction buffers for cleaner preparations

• Technical validation approaches:

  • Run peptide competition controls alongside experimental samples

  • Include knockout/knockdown samples to identify specific bands

  • Use gradient gels for better separation of similar molecular weight proteins

  • Implement membrane stripping and reprobing protocols to confirm specificity

These systematic approaches will help researchers distinguish specific ABCI7 signal from non-specific background, enhancing experimental reliability and reproducibility.

What are the best approaches for quantifying relative ABCI7 protein levels across different experimental conditions?

Accurate quantification of ABCI7 protein levels requires careful methodological consideration:

• Western blot quantification best practices:

  • Use chemiluminescent detection within linear range

  • Alternatively, employ fluorescent secondary antibodies for wider linear range

  • Include standard curve with purified recombinant protein

  • Normalize to multiple housekeeping proteins (actin, GAPDH, tubulin)

  • Process all experimental conditions on the same blot when possible

• ELISA-based quantification approach:

  • Develop sandwich ELISA using capture and detection antibodies

  • Include standard curve with known quantities of recombinant ABCI7

  • Run all samples in technical triplicates

  • Use four-parameter logistic regression for standard curve fitting

  • Calculate concentration based on standard curve interpolation

• Data normalization strategies:

  • Total protein normalization using Ponceau S or Coomassie staining

  • Housekeeping protein ratio analysis

  • Geometric mean of multiple reference proteins

  • Global normalization using total signal intensity

• Statistical analysis framework:

  • Apply appropriate statistical tests based on experimental design

  • Calculate confidence intervals for all measurements

  • Report both raw and normalized values

  • Use power analysis to determine required sample sizes

How can researchers design validation experiments to confirm ABCI7 antibody specificity in plant systems?

• Genetic validation approaches:

  • Test antibody in ABCI7 knockout/knockdown lines (T-DNA insertion, CRISPR/Cas9)

  • Perform complementation studies with tagged ABCI7 constructs

  • Use RNAi lines with varying degrees of ABCI7 suppression

  • Compare antibody signal with mRNA expression patterns

• Biochemical validation methods:

  • Perform peptide competition assays using immunizing peptide

  • Conduct immunoprecipitation followed by mass spectrometry

  • Compare multiple antibodies targeting different ABCI7 epitopes

  • Test cross-reactivity with recombinant related proteins (other ABC transporters)

• Heterologous expression validation:

  • Express tagged ABCI7 in heterologous systems (E. coli, yeast)

  • Compare detection of native vs. heterologously expressed protein

  • Create fusion proteins with different tags for orthogonal detection

  • Test antibody performance across expression levels

• Technical controls implementation:

  • Include pre-immune serum controls in all experiments

  • Perform secondary-only controls to assess non-specific binding

  • Use isotype controls at equivalent concentrations

  • Implement peptide blocking controls systematically

How can ABCI7 antibody be adapted for use in chromatin immunoprecipitation (ChIP) experiments to study potential DNA-binding activities?

While ABCI7 is primarily characterized as a transport protein, investigating potential DNA interactions requires specialized ChIP adaptations:

• ChIP protocol optimization for ABCI7:

  • Crosslinking optimization (1-3% formaldehyde for varying times)

  • Sonication parameters adjustment for plant chromatin (power, cycles)

  • Antibody concentration titration (2-10 μg per reaction)

  • Enrichment verification using known DNA-binding proteins as positive controls

• Controls for ChIP specificity:

  • IgG negative control processed in parallel

  • Input chromatin control for normalization

  • ABCI7 knockout/knockdown negative control

  • Positive control using known DNA-binding transcription factors

• Analysis approaches:

  • qPCR for specific target regions

  • ChIP-seq for genome-wide binding profile

  • De novo motif discovery in enriched regions

  • Integration with transcriptome data for functional correlation

• Validation strategies:

  • Electrophoretic mobility shift assay (EMSA) with recombinant ABCI7

  • In vitro DNA-binding assays with purified protein

  • Reporter gene assays for functional validation

  • Protein-DNA crosslinking followed by mass spectrometry

These methodological considerations enable investigation of potential novel functions of ABCI7 beyond its characterized role in transport processes.

What considerations should researchers address when using ABCI7 antibody for super-resolution microscopy techniques?

Super-resolution microscopy offers nanoscale visualization of protein distribution and requires specific antibody considerations:

• Fixation and sample preparation optimization:

  • Test aldehyde-based fixatives versus organic solvent fixation

  • Optimize cell wall digestion for improved antibody penetration

  • Evaluate clearing methods for deep tissue imaging

  • Balance fixation strength with epitope preservation

• Antibody labeling strategies:

  • Direct conjugation with small fluorophores (Alexa Fluor 647, Cy5)

  • Use of smaller detection probes (nanobodies, aptamers)

  • Two-step labeling with biotinylated primary and fluorophore-streptavidin

  • Evaluation of quantum dots for PALM/STORM applications

• Validation controls:

  • Correlation with diffraction-limited conventional microscopy

  • Parallel imaging of fluorescent protein fusions

  • Quantification of labeling density and specificity

  • Background assessment in knockout/knockdown samples

• Technical considerations for different super-resolution techniques:

Implementing these considerations enables nanoscale visualization of ABCI7 distribution and potential co-localization with interaction partners.

How can researchers integrate ABCI7 antibody-based assays with functional transport studies to correlate expression with activity?

Correlating ABCI7 protein levels with transport function requires integrated experimental approaches:

• Parallel quantification strategies:

  • Quantitative Western blot for protein expression level

  • Transport assays in isolated membrane vesicles

  • ATP hydrolysis measurements as proxy for transport activity

  • Development of substrate accumulation assays in intact organelles

• Genetic manipulation approaches:

  • Create expression gradient using inducible promoters

  • Generate point mutations affecting transport but not stability

  • Implement tissue-specific or developmentally regulated expression

  • Use CRISPR/Cas9 for precise genome editing

• Correlation analysis framework:

  • Plot protein expression vs. transport activity

  • Calculate Pearson/Spearman correlation coefficients

  • Determine threshold expression for detectable activity

  • Model relationship between expression and function

• Integrated experimental design:

  • Time-course analysis following induction or repression

  • Environmental stress response with parallel protein/activity measurements

  • In vitro reconstitution with defined protein amounts

  • Single-cell analysis correlating expression with functional readouts

This integrated approach provides mechanistic insight into the relationship between ABCI7 expression levels and its functional transport activity, revealing potential regulatory mechanisms.

What experimental designs can address potential post-translational modifications of ABCI7 using the available antibody?

Investigation of post-translational modifications (PTMs) requires specialized approaches:

• Detection of common PTMs:

  • Phosphorylation: Phos-tag gels, phospho-specific antibodies

  • Ubiquitination: Immunoprecipitation under denaturing conditions

  • Glycosylation: Glycosidase treatment, lectin blotting

  • Acetylation: Acetylation-specific antibodies, deacetylase inhibitors

• Mass spectrometry-based approaches:

  • Immunoprecipitation followed by LC-MS/MS

  • Enrichment of specific modifications (TiO₂ for phosphopeptides)

  • Targeted multiple reaction monitoring (MRM) for specific sites

  • Quantitative comparison across conditions

• Functional correlation studies:

  • Site-directed mutagenesis of predicted modification sites

  • Treatment with modification-inducing conditions

  • Inhibitor studies targeting specific modification enzymes

  • Correlation of modification status with protein activity

• Visualization of modification dynamics:

  • Development of PTM-specific probes

  • Time-course analysis following stimulus

  • Subcellular fractionation to track modified pools

  • Co-immunoprecipitation to identify modification-dependent interactions

These approaches enable comprehensive characterization of ABCI7 post-translational modifications and their functional significance in plant physiology.

How does ABCI7 structure and function compare with other ATP-binding cassette transporters in plants?

Comparative analysis of ABCI7 with other ABC transporters provides evolutionary and functional context:

• Structural comparison approaches:

  • Domain architecture analysis (transmembrane domains vs. nucleotide-binding domains)

  • Homology modeling based on crystallized ABC transporters

  • Sequence conservation mapping to functional regions

  • Phylogenetic analysis of ABC transporter subfamilies

• Functional comparison strategies:

  • Substrate specificity profiling across ABC transporters

  • ATP hydrolysis kinetics comparison

  • Transport mechanism evaluation (conformational changes)

  • Regulation pattern analysis across different conditions

• Experimental comparison framework:

  • Parallel phenotypic analysis of knockout lines

  • Cross-complementation studies between transporters

  • Side-by-side biochemical characterization

  • Comparative protein interaction network analysis

ABCI7 belongs to a specialized subset of ATP-binding cassette proteins with distinct structural features compared to full-size ABC transporters. It lacks transmembrane domains typical of many transporters and likely functions as part of larger protein complexes.

While mammalian ABCB7 exports iron-sulfur clusters from mitochondria to cytosol , plant ABCI7 may have evolved different substrate specificities and cellular functions related to plant-specific metabolic and developmental processes.