At4g19060 Antibody

What is the At4g19060 gene and what protein does it encode?

At4g19060 is an Arabidopsis thaliana gene that encodes ACBP6, a 10.4-kDa acyl-coenzyme A binding protein. Unlike other members of the Arabidopsis ACBP family (which ranges from 37.5 to 73.1 kD), ACBP6 is the smallest member and functions primarily in the cytosol . ACBP6 has been shown to play significant roles in phospholipid metabolism and cold stress responses in plants. The protein consists of approximately 92 amino acids and contains a highly conserved acyl-CoA-binding domain that facilitates interaction with long-chain acyl-CoA esters .

What is the subcellular localization of the ACBP6 protein?

ACBP6 is primarily localized in the cytosol of plant cells. This localization has been confirmed through multiple experimental approaches:

• Analysis of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 (ACBP6-GFP)

• Western-blot analysis of subcellular fractions using ACBP6-specific antibodies

When subcellular fractions were analyzed by western blotting, the 10.4-kD ACBP6 band was detected in the total protein and cytosolic fractions, but was absent in membrane, organelle (mitochondria, chloroplasts, peroxisomes), and nuclear fractions . While some ACBP6-GFP signals were observed in nuclei in transgenic plants, this was determined to be due to diffusion of the fusion protein rather than targeted localization, as ACBP6 lacks a nuclear targeting signal .

How can I validate the specificity of an At4g19060/ACBP6 antibody?

Validation of an ACBP6 antibody should include the following approaches:

• Western blot analysis using wild-type vs. knockout plants: Compare protein extracts from wild-type Arabidopsis and acbp6 knockout mutants. A specific antibody will detect a 10.4-kD band in wild-type samples that is absent in the knockout .

• Protein expression verification: Test the antibody against purified recombinant ACBP6 protein as a positive control.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other ACBP family members by testing against protein extracts from plants overexpressing different ACBP proteins.

• Subcellular fractionation: Confirm that the antibody detects ACBP6 predominantly in cytosolic fractions .

• Immunoprecipitation validation: Verify that the antibody can specifically immunoprecipitate ACBP6 from plant extracts.

How can ACBP6 antibodies be used to study cold stress responses in plants?

ACBP6 antibodies are valuable tools for studying cold stress responses because ACBP6 expression is significantly induced by cold treatment. Research has demonstrated that:

• ACBP6 protein accumulation peaks at 48 hours following exposure to 4°C, as detected by western blot analysis .

• This cold-induced expression pattern correlates with ACBP6's role in freezing tolerance.

Experimental applications include:

• Time-course studies: Monitor ACBP6 protein levels at different timepoints (0, 6, 12, 24, and 48 hours) after cold treatment using western blot analysis .

• Comparative analysis: Compare ACBP6 protein levels across different genetic backgrounds (wild-type, acbp6 mutants, and ACBP6-overexpressing lines) under cold stress.

• Co-immunoprecipitation: Identify cold-responsive protein interaction partners using ACBP6 antibodies for pull-down experiments.

Table 1: Temporal pattern of ACBP6 protein accumulation during cold treatment based on western blot quantification data from Arabidopsis thaliana plants .

What methods can be used to optimize immunodetection of ACBP6 in plant tissues?

Optimizing ACBP6 immunodetection requires specific considerations due to its small size (10.4 kDa) and cytosolic localization:

• Sample preparation: Use a protein extraction buffer optimized for cytosolic proteins (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, with protease inhibitors).

• Gel electrophoresis conditions: Employ higher percentage (15-18%) SDS-PAGE gels to properly resolve small proteins like ACBP6.

• Transfer optimization: Use specialized transfer conditions for small proteins:

  • PVDF membranes with 0.2 μm pore size (instead of 0.45 μm)

  • Transfer buffer with 20% methanol

  • Lower voltage transfer (30V) for longer duration (2 hours)

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBS-T, with optimization to reduce background while maintaining specific signals.

• Antibody dilution: Initial testing should include a dilution series (1:500 to 1:5000) to determine optimal concentration for specific detection.

• Enhanced detection systems: Consider using high-sensitivity chemiluminescent substrates for detection of low-abundance cytosolic proteins.

How can ACBP6 antibodies be used to investigate protein-lipid interactions?

ACBP6 has been demonstrated to bind phosphatidylcholine but not phosphatidic acid or lysophosphatidylcholine in in vitro filter-binding assays . Researchers can leverage ACBP6 antibodies to explore these interactions through several sophisticated approaches:

• Immunoprecipitation-coupled lipid analysis: Use ACBP6 antibodies to immunoprecipitate the protein along with bound lipids, followed by lipid extraction and analysis by thin-layer chromatography or mass spectrometry.

• Proximity ligation assays: Combine ACBP6 antibodies with lipid-binding probes to visualize protein-lipid interactions in situ using fluorescence microscopy.

• Co-sedimentation assays: Utilize ACBP6 antibodies to detect the protein in liposome binding experiments to quantify affinities for different phospholipids.

• Cross-linking mass spectrometry: Identify specific amino acid residues involved in lipid binding by cross-linking ACBP6 with its lipid partners followed by immunoprecipitation and mass spectrometry analysis.

Research findings indicate that ACBP6 plays a role in phospholipid metabolism, particularly in cold stress conditions, where overexpression leads to:

• 36-46% decrease in phosphatidylcholine

• 67-73% increase in phosphatidic acid levels in cold-acclimated, freezing-treated plants

What approaches can be used to study ACBP6 involvement in phospholipid metabolism during cold stress?

ACBP6 antibodies can be employed in multiple experimental paradigms to investigate its role in cold-induced changes in phospholipid metabolism:

• Quantitative western blotting: Compare ACBP6 protein levels with changes in phospholipid profiles during cold acclimation and freezing treatment.

• Immunolocalization studies: Track potential redistribution of ACBP6 within the cytosol during cold stress using immunofluorescence microscopy.

• Protein-protein interaction analysis: Identify interactions between ACBP6 and enzymes involved in phospholipid metabolism (such as phospholipase D) during cold stress using co-immunoprecipitation with ACBP6 antibodies.

• Differential extraction experiments: Analyze whether cold stress affects the association of ACBP6 with membrane fractions by sequential extraction followed by immunoblotting.

Table 2: Phospholipid composition changes in wild-type versus ACBP6-overexpressing Arabidopsis plants after cold acclimation and freezing treatment (-8°C) .

What are the optimal antigenic regions of ACBP6 for antibody production?

When developing antibodies against ACBP6, careful consideration of antigenic regions is crucial due to the protein's small size and the presence of a highly conserved acyl-CoA-binding domain:

• Epitope selection strategies:

  • Target unique regions that distinguish ACBP6 from other ACBP family members

  • Avoid the highly conserved acyl-CoA-binding pocket to ensure specificity

  • Consider the C-terminal region (amino acids 70-92) which typically contains more variable sequences

• Peptide design considerations:

  • Optimal peptide length: 15-20 amino acids

  • Ensure surface accessibility (use hydrophilicity prediction)

  • Verify uniqueness through BLAST analysis against the Arabidopsis proteome

  • Consider coupling the peptide to carrier proteins like KLH or BSA for immunization

• Full-length protein versus peptide approaches:

  • Recombinant full-length ACBP6 can provide antibodies against multiple epitopes

  • Peptide-based approaches allow targeting of specific regions but may yield lower titer antibodies

How can reporter systems be used to validate ACBP6 antibody specificity in vivo?

Several sophisticated reporter systems can be employed to validate ACBP6 antibody specificity in living plant systems:

• Fluorescent protein fusion validation:

  • Generate transgenic plants expressing ACBP6-GFP fusion proteins

  • Perform western blots using both anti-GFP and anti-ACBP6 antibodies on the same samples

  • Co-localization of signals confirms antibody specificity

• Inducible expression systems:

  • Develop transgenic lines with estrogen- or dexamethasone-inducible ACBP6 expression

  • Compare antibody detection before and after induction

  • Quantitative correlation between induction level and antibody signal strength validates specificity

• CRISPR/Cas9 epitope tagging:

  • Use genome editing to add small epitope tags to endogenous ACBP6

  • Compare detection patterns between ACBP6 antibody and commercial tag antibodies

  • Identical patterns confirm specificity

How does ACBP6 function differ from other Arabidopsis ACBP family members in stress responses?

ACBP6 exhibits unique functions compared to other ACBP family members in Arabidopsis, particularly in the context of stress responses:

• Differential expression patterns:

  • ACBP6 shows strong induction at 48 hours after cold treatment

  • Other family members may show different temporal expression patterns or respond to different stressors

• Functional specialization:

  • ACBP6 is involved in phospholipid metabolism during cold stress

  • Other ACBPs may have roles in different aspects of lipid metabolism or stress responses

• Protein interaction networks:

  • ACBP6-specific antibodies can be used in comparative co-immunoprecipitation studies to identify unique interaction partners for each family member

  • Differential interactomes may explain functional specialization

Researchers can use ACBP6 antibodies alongside antibodies against other ACBP family members to perform comparative studies that illuminate the specific roles of each protein in stress responses.

How can ACBP6 antibodies be used to investigate cross-talk between cold stress and other environmental stresses?

ACBP6 antibodies provide valuable tools for studying potential overlap between cold stress and other environmental stress responses:

• Multi-stress experimental design:

  • Expose plants to combined stresses (cold + drought, cold + salt, etc.)

  • Use ACBP6 antibodies to monitor protein expression patterns

  • Compare with single-stress treatments to identify synergistic or antagonistic effects

• Hormone treatment studies:

  • Treat plants with stress hormones (ABA, ethylene, jasmonic acid) before or during cold exposure

  • Monitor ACBP6 protein levels to identify potential regulatory crosstalk

• Chromatin immunoprecipitation (ChIP) studies:

  • Use antibodies against transcription factors involved in different stress pathways

  • Identify potential binding to ACBP6 promoter regions under various stress conditions

• Protein modification analysis:

  • Investigate potential post-translational modifications of ACBP6 under different stress conditions

  • Phosphorylation or other modifications may reveal integration of multiple stress signaling pathways

Table 3: Comparative ACBP6 protein expression under different stress conditions based on western blot quantification .

What immunohistochemical techniques are most effective for localizing ACBP6 in plant tissues?

Due to ACBP6's cytosolic localization and relatively small size, specialized immunohistochemical approaches are recommended:

• Tissue preparation options:

  • Chemical fixation: 4% paraformaldehyde with 0.1% glutaraldehyde preserves cytosolic proteins while maintaining antigenicity

  • Cryofixation: Rapid freezing followed by freeze-substitution preserves native protein localization

• Sectioning approaches:

  • Vibratome sections (50-100 μm) for preliminary localization studies

  • Thin resin sections (1-2 μm) for higher resolution imaging

  • Cryosections for preservation of antigenicity

• Signal amplification methods:

  • Tyramide signal amplification for detection of low-abundance proteins

  • Quantum dot-conjugated secondary antibodies for improved sensitivity and stability

• Counterstaining strategies:

  • DAPI for nuclear visualization

  • FM4-64 for membrane counterstaining

  • Combination with other organelle-specific antibodies for co-localization studies

• Controls and validation:

  • Include acbp6 knockout tissues as negative controls

  • Use ACBP6-GFP transgenic lines as positive controls

  • Perform peptide competition assays to confirm specificity

How can proximity-dependent labeling techniques be combined with ACBP6 antibodies to identify novel interaction partners?

Proximity-dependent labeling represents an advanced approach for identifying proteins that interact with ACBP6 in vivo:

• BioID methodology:

  • Generate transgenic plants expressing ACBP6-BirA* fusion protein

  • After biotin treatment, biotinylated proteins in close proximity to ACBP6 can be purified

  • Validate using ACBP6 antibodies to confirm expression of the fusion protein

• APEX2 proximity labeling:

  • Express ACBP6-APEX2 fusion in plants

  • After brief H₂O₂ treatment, proteins near ACBP6 become biotinylated

  • Compare biotinylated protein profiles with ACBP6 immunoprecipitation results

• Split-BioID approach:

  • Fuse ACBP6 with one half of split-BirA

  • Fuse candidate interactors with complementary BirA half

  • Interaction brings BirA halves together, enabling proximity labeling

  • Validate interactions using co-immunoprecipitation with ACBP6 antibodies

• Comparative interactome analysis:

  • Generate interactome maps under control versus stress conditions

  • Identify stress-specific interaction partners

  • Validate key interactions using reciprocal co-immunoprecipitation with ACBP6 antibodies