At5g07830 Antibody

What is the At5g07830 gene and what protein does it encode?

At5g07830 is the Arabidopsis thaliana gene locus that encodes ACBP6, a 10-kilodalton acyl-CoA binding protein. ACBP6 is the smallest member of the Arabidopsis ACBP family, which includes six members ranging from 10.4 kD to 73.1 kD . Unlike its larger family members (ACBP1-5), ACBP6 is primarily cytosolic and has well-characterized homologs in other eukaryotes . The protein functions in binding and transport of acyl-CoA esters and potentially in gene regulation, similar to its mammalian counterparts .

How can I confirm the specificity of an At5g07830/ACBP6 antibody?

Confirming antibody specificity for ACBP6 requires multiple validation approaches:

• Western blot analysis using both wild-type plants and acbp6 knockout mutants to demonstrate absence of the signal in the mutant

• Protein expression verification by comparing results with northern blot data showing mRNA expression

• Testing for cross-reactivity with other ACBP family members (ACBP1-5) using recombinant proteins

• Subcellular fractionation followed by western blot to confirm the expected cytosolic localization pattern

When analyzing western blots, a specific ACBP6 antibody should detect a band at approximately 10.4 kD in wild-type plants but not in acbp6 knockout mutants . Additionally, the antibody should primarily detect the protein in cytosolic fractions with minimal signal in membrane fractions.

What are the optimal conditions for At5g07830/ACBP6 antibody use in western blotting?

For optimal western blot results with ACBP6 antibodies:

• Sample preparation: Extract total protein from plant tissues in buffer containing protease inhibitors

• Protein separation: Use 15-18% SDS-PAGE gels due to the small size (10.4 kD) of ACBP6

• Transfer: Employ PVDF membranes with small pore size and optimize transfer time for small proteins

• Blocking: 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature

• Primary antibody incubation: Dilute ACBP6-specific antibody (typically 1:1000 to 1:5000) and incubate overnight at 4°C

• Detection: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL)

When analyzing stress-induced expression, such as cold treatment, extend the analysis to multiple time points (e.g., 24h, 48h, 72h) as ACBP6 shows noticeably induced expression at 48h after 4°C treatment .

What controls should I include when using At5g07830/ACBP6 antibodies?

Robust experimental design for ACBP6 antibody applications should include:

• Positive control: Recombinant ACBP6 protein or extract from ACBP6-overexpressing plants

• Negative control: Extract from acbp6 T-DNA insertional mutant plants

• Loading control: Antibody against a constitutively expressed cytosolic protein (e.g., GAPDH)

• Cross-reactivity control: Extracts from plants overexpressing other ACBP family members

• Subcellular marker controls: When performing localization studies, include antibodies against known subcellular compartment markers (e.g., histone H3 for nuclei, BiP for ER)

For immunolocalization experiments, additional controls should include pre-immune serum and primary antibody omission controls to assess non-specific binding.

How do I design experiments to detect stress-induced changes in At5g07830/ACBP6 expression?

Designing experiments to detect stress-induced changes in ACBP6 expression requires:

• Stress treatment design:

  • Cold stress: Expose plants to 4°C for varying durations (0, 24, 48, 72 hours)

  • Include multiple stress conditions (drought, salt, heat) for comparative analysis

• Multi-level analysis approach:

  • Transcriptional level: Northern blot or RT-qPCR analysis of ACBP6 mRNA expression

  • Protein level: Western blot with ACBP6-specific antibodies

  • Promoter activity: Generate ACBP6 promoter-reporter constructs

• Sampling strategy:

  • Collect samples at defined intervals (e.g., 0, 6, 12, 24, 48, 72 hours after treatment)

  • Separate analysis of different plant tissues (roots, leaves, stems)

Research has demonstrated that ACBP6 expression is notably induced at 48h after 4°C treatment, as confirmed by both northern blot and western blot analysis . This temporal expression pattern suggests a role in cold stress adaptation that can be further investigated through antibody-based approaches.

How can I use At5g07830/ACBP6 antibodies to investigate protein-protein interactions?

To investigate ACBP6 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use ACBP6 antibodies immobilized on protein A/G beads

  • Incubate with plant extracts under non-denaturing conditions

  • Elute and analyze precipitated complexes by mass spectrometry

  • Validate interactions with western blot using antibodies against suspected interacting partners

• Proximity labeling approaches:

  • Generate ACBP6 fusion with BioID or APEX2

  • Identify proteins in close proximity through biotinylation

  • Validate with ACBP6 antibodies in reciprocal Co-IP

• Pull-down assays:

  • Express recombinant ACBP6 with affinity tag

  • Validate pulled-down complexes with ACBP6 antibodies

  • Compare results with native immunoprecipitation using ACBP6 antibodies

When designing these experiments, consider that ACBP6 has been detected in both cytosolic and nuclear fractions , suggesting potential interactions with both cytosolic proteins and nuclear factors, similar to its mammalian counterparts that interact with nuclear factor-4α .

What are the best practices for using At5g07830/ACBP6 antibodies in immunolocalization studies?

For optimal immunolocalization of ACBP6:

• Tissue preparation:

  • Fix tissues with 4% paraformaldehyde

  • Consider both paraffin embedding and cryosectioning approaches

  • For whole-mount preparations, optimize cell wall permeabilization

• Antibody validation:

  • Compare antibody localization with ACBP6-GFP fluorescence patterns

  • Include acbp6 mutant tissues as negative controls

  • Use pre-immune serum controls

• Dual/multi-labeling:

  • Combine ACBP6 antibodies with markers for subcellular compartments

  • Use fluorophore-conjugated secondary antibodies with non-overlapping spectra

  • Include nuclear counterstains (e.g., DAPI)

Research has confirmed that ACBP6 is primarily localized to the cytosol with some signals in the nuclei, as demonstrated by both ACBP6-GFP fusion studies and western blot analysis of subcellular fractions using ACBP6-specific antibodies . This dual localization pattern should be considered when interpreting immunolocalization results.

How do I design experiments to investigate the relationship between At5g07830/ACBP6 and lipid metabolism using antibodies?

To investigate ACBP6's role in lipid metabolism:

• Expression correlation studies:

  • Monitor ACBP6 protein levels using antibodies alongside lipid profile changes

  • Compare wild-type, acbp6 knockout, and ACBP6-overexpressing plants

  • Analyze under normal and stress conditions (particularly cold stress)

• Subcellular analysis:

  • Fractionate cells to isolate cytosol, nuclei, and membrane compartments

  • Use ACBP6 antibodies to quantify protein distribution

  • Correlate with lipid composition of each fraction

• Immunoprecipitation of lipid-protein complexes:

  • Use ACBP6 antibodies to isolate protein from plant extracts

  • Analyze co-precipitated lipids by mass spectrometry

  • Compare lipid profiles between wild-type and stress-induced plants

• In vitro binding assays:

  • Combine recombinant ACBP6 with various acyl-CoA esters

  • Use antibodies to pull down protein-lipid complexes

  • Quantify binding affinity and specificity

Consider that ACBP6, as a 10-kD acyl-CoA binding protein, is likely involved in binding and transport of cytosolic acyl-CoA esters, similar to its mammalian homologs . The cold-inducible expression pattern suggests potential roles in membrane lipid remodeling during temperature stress.

What approaches can be used to investigate the functional redundancy between ACBP6 and other ACBP family members using antibodies?

To investigate functional redundancy among ACBP family members:

• Expression analysis in mutant backgrounds:

  • Generate single, double, and higher-order acbp mutants

  • Use antibodies specific to each ACBP to check for compensatory expression

  • Quantify protein levels in different tissues and developmental stages

• Subcellular distribution comparison:

  • Use specific antibodies against each ACBP family member

  • Compare localization patterns in subcellular fractions

  • Analyze changes in localization in various mutant backgrounds

• Protein-protein interaction network mapping:

  • Perform immunoprecipitation with antibodies against each ACBP

  • Identify unique and overlapping interacting partners

  • Construct interaction networks to identify common pathways

• Stress response profiling:

  • Monitor protein levels of all ACBPs during stress using specific antibodies

  • Compare expression timing and tissue distribution

  • Correlate with physiological responses in various mutant combinations

Remember that Arabidopsis contains six ACBP forms (ACBP1-6) ranging from 10.4 kD to 73.1 kD, with diverse subcellular localizations: ACBP1 and ACBP2 are membrane-associated (ER and plasma membrane), ACBP3 is extracellularly targeted, ACBP4 and ACBP5 contain Kelch motifs, while only ACBP6 is primarily cytosolic . This diverse localization suggests specialized functions despite potential redundancy.

Why might I detect multiple bands when using At5g07830/ACBP6 antibodies in western blots?

Multiple bands in ACBP6 western blots may result from:

• Post-translational modifications:

  • Phosphorylation (adds ~80 Da per phosphate group)

  • Ubiquitination (adds ~8.5 kDa per ubiquitin)

  • Other modifications affecting protein mobility

• Technical issues:

  • Sample degradation during preparation

  • Incomplete denaturation causing oligomerization

  • Non-specific antibody binding to other ACBP family members

• Protein isoforms:

  • Alternative splicing variants

  • Proteolytic processing

  • Different start codon usage

Troubleshooting steps include:

• Compare observed band patterns with theoretical molecular weight (10.4 kD for ACBP6)

• Include both acbp6 knockout and ACBP6-overexpressing samples

• Pre-absorb antibody with recombinant ACBP6 protein

• Add phosphatase inhibitors to test for phosphorylation-dependent bands

• Optimize sample preparation and gel running conditions

When analyzing ACBP6-GFP fusion proteins, expect a band at approximately 38.4 kD (consisting of 10.4 kD ACBP6 fused to 28 kD GFP) .

How can I optimize At5g07830/ACBP6 antibody performance for detecting low abundance protein?

To optimize detection of low-abundance ACBP6:

• Sample enrichment methods:

  • Concentrate cytosolic fractions where ACBP6 primarily localizes

  • Use immunoprecipitation to enrich before western blotting

  • Consider tissues/conditions with higher expression (cold-treated for 48h)

• Signal amplification techniques:

  • Use high-sensitivity ECL substrates

  • Consider tyramide signal amplification for immunohistochemistry

  • Try biotin-streptavidin detection systems

• Antibody optimization:

  • Test different antibody concentrations (titration series)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use monoclonal antibodies for higher specificity

• Reduce background:

  • Optimize blocking conditions (test BSA vs. milk)

  • Include 0.1-0.3% Triton X-100 in antibody diluent

  • Increase washing duration and number of washes

Consider that ACBP6 expression is induced under certain stress conditions, particularly cold treatment (4°C for 48h) , which can be utilized to obtain samples with higher protein abundance for initial optimization.

How do At5g07830/ACBP6 antibodies compare with antibodies against homologous proteins from other species?

When comparing ACBP6 antibodies with those against homologs from other species:

• Cross-reactivity analysis:

  • Test Arabidopsis ACBP6 antibodies against proteins from other plant species

  • Compare epitope conservation across species using sequence alignment

  • Validate cross-reactive antibodies through western blot with heterologous proteins

• Functional conservation assessment:

  • Use antibodies to compare subcellular localization across species

  • Compare expression patterns under similar stress conditions

  • Investigate if proteins recognize similar interacting partners

• Evolutionary implications:

  • Use antibodies to track protein abundance across evolutionary diverse plants

  • Compare post-translational modifications between species

  • Evaluate structural conservation through epitope mapping

The 10-kD ACBP6 is notable for having well-characterized homologs in other eukaryotes, unlike the larger ACBP1-5 family members . This conservation suggests fundamental roles that may be studied comparatively using antibodies with confirmed cross-reactivity.

How can I use At5g07830/ACBP6 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For ChIP experiments with ACBP6 antibodies:

• Experimental design considerations:

  • Optimize crosslinking conditions (typically 1% formaldehyde, 10-15 minutes)

  • Include appropriate controls (IgG, input, non-crosslinked samples)

  • Consider nuclear enrichment before immunoprecipitation

• Protocol optimization:

  • Test different sonication conditions to generate 200-500 bp fragments

  • Optimize antibody concentration and incubation conditions

  • Include controls for non-specific binding

• Results interpretation:

  • Validate enriched regions with qPCR before sequencing

  • Compare binding patterns under different stress conditions

  • Correlate with transcriptional changes of target genes

• Validation approaches:

  • Confirm binding with electrophoretic mobility shift assays

  • Use reporter gene assays to test functional significance

  • Perform reciprocal experiments with suspected transcription factor partners

ACBP6 has been detected in both cytosolic and nuclear fractions , and mammalian ACBP homologs interact with nuclear transcription factors , suggesting potential roles in transcriptional regulation that can be investigated through ChIP approaches.

What future research directions might require At5g07830/ACBP6 antibodies?

Promising future research using ACBP6 antibodies includes:

• Systems biology approaches:

  • Proteome-wide interaction mapping using antibody-based techniques

  • Spatial and temporal profiling of ACBP6 abundance during development

  • Integration with metabolomics to correlate protein levels with lipid profiles

• Stress adaptation mechanisms:

  • Detailed characterization of cold-responsive expression patterns

  • Investigation of ACBP6 roles in membrane lipid remodeling during temperature fluctuations

  • Comparative analysis across species with different cold tolerance

• Translational applications:

  • Engineering stress tolerance through modulation of ACBP6 expression

  • Developing ACBP6-based biosensors for monitoring cellular lipid metabolism

  • Exploring biotechnological applications in improving crop stress resilience

• Structural biology integration:

  • Using antibodies to capture different conformational states

  • Investigating protein-protein and protein-lipid interactions at molecular level

  • Correlating structural features with functional outcomes