At5g10340 Antibody

What is At5g10340 and why is an antibody against it valuable for plant research?

At5g10340 is the gene ID for ACBP6, a 10-kilodalton acyl-coenzyme A binding protein in Arabidopsis thaliana. An antibody against this protein is valuable for studying its expression patterns, subcellular localization, and roles in stress responses. Research has shown that ACBP6 expression is notably induced after 48 hours of exposure to 4°C, making it an important target for investigating plant cold acclimation mechanisms . The antibody enables detection of this protein through various experimental applications, providing insights into lipid metabolism and stress adaptation in plants.

How can researchers confirm the specificity of an At5g10340/ACBP6 antibody?

Confirming antibody specificity requires multiple validation approaches. Western blot analysis should be performed using wild-type Arabidopsis samples alongside acbp6 knockout mutants to verify absence of signal in mutants. Subcellular fractionation followed by immunoblotting can confirm the expected cytosolic localization pattern of the 10.4-kD ACBP6 protein . Researchers should implement direct binding assays with both positive controls (purified ACBP6 protein) and negative controls (irrelevant isotype-matched antibodies) . Cross-reactivity testing against other ACBP family members is essential, as Arabidopsis contains multiple ACBP proteins with varying sizes (37.5 to 73.1 kD) . Finally, peptide competition assays can further validate specificity by demonstrating signal reduction when the antibody is pre-incubated with the target antigen.

What are the key quality control parameters for At5g10340/ACBP6 antibody production?

Quality control for ACBP6 antibodies should include comprehensive characterization of structural integrity, specificity, and potency. Structural integrity should be assessed through a combination of SDS-PAGE, IEF, HPLC, and/or mass spectrometry to confirm the antibody is not fragmented or aggregated . Specificity verification requires side-by-side comparisons with qualified reference standards and testing against both the target protein and related ACBP family members . Potency assays should quantitatively measure binding activity through affinity and avidity assessments . Additional quality control measures include sterility testing, endotoxin measurement, and lot-to-lot consistency evaluation. Researchers should establish a properly qualified in-house reference standard stored under appropriate conditions for consistent comparisons between production batches .

What is the optimal protocol for using At5g10340/ACBP6 antibody in Western blot analysis?

For optimal Western blot analysis of ACBP6, researchers should carefully consider sample preparation and gel conditions. Since ACBP6 is a small 10.4-kD protein, use 15-18% polyacrylamide gels for proper resolution . Extract proteins using buffers containing protease inhibitors to prevent degradation. For efficient transfer of this small protein, PVDF membranes and methanol-containing transfer buffers are recommended. Blocking should be performed with 5% non-fat milk or BSA in TBST. Primary antibody dilution should be optimized through titration experiments, typically starting at 1:1000. Essential controls include positive controls (recombinant ACBP6), negative controls (acbp6 knockout extracts), and loading controls. For subcellular localization studies, perform cellular fractionation prior to Western blotting, as previous research has successfully detected native ACBP6 in cytosolic fractions but not in membrane, organellar, or nuclear fractions of Arabidopsis .

How can researchers use At5g10340/ACBP6 antibody to study protein expression changes during stress responses?

To study ACBP6 expression changes during stress responses, implement time-course experiments collecting samples at multiple timepoints after stress treatment. Research has shown ACBP6 induction 48 hours after cold treatment using both northern-blot and western-blot analysis . For quantitative assessment, perform Western blots with internal loading controls and analyze band intensity using densitometry software. Normalize ACBP6 signal to loading controls and calculate relative expression compared to untreated samples. Include appropriate time-matched controls to account for developmental or circadian effects. For comprehensive analysis, combine protein-level detection using the antibody with transcript-level assessment via RT-qPCR. This approach allows researchers to determine whether expression changes occur at transcriptional or post-transcriptional levels. Additionally, examine potential stress-induced changes in subcellular distribution by performing fractionation studies under control and stress conditions.

What approaches are recommended for immunolocalization of At5g10340/ACBP6 in plant tissues?

For immunolocalization of ACBP6, researchers should implement both antibody-based detection and complementary approaches. For tissue sections, fix samples in paraformaldehyde, embed in appropriate medium, section, and perform immunostaining with the ACBP6 antibody followed by fluorescently-labeled secondary antibodies. For subcellular localization, perform differential centrifugation to separate cellular components, then analyze fractions by Western blotting . Complement antibody-based approaches with fluorescent protein fusions; previous research confirmed cytosolic localization of ACBP6 through both antibody detection in subcellular fractions and observation of ACBP6-GFP fusion proteins . Essential controls include acbp6 knockout tissues as negative controls, pre-immune serum controls, and peptide competition assays. For co-localization studies, combine ACBP6 antibody with markers for different cellular compartments. When using ACBP6-GFP fusions, compare results with antibody-based detection to confirm that tagging doesn't alter localization patterns.

How can researchers address cross-reactivity issues with At5g10340/ACBP6 antibody?

Cross-reactivity issues with ACBP6 antibody can be addressed through several targeted approaches. First, perform epitope mapping to identify specific regions of ACBP6 recognized by the antibody and compare with homologous regions in other ACBPs. Consider affinity purification by passing the antibody through columns containing immobilized related proteins to deplete cross-reactive antibodies. Implement absorption controls by pre-incubating the antibody with excess recombinant related proteins before use. Utilize genetic controls such as knockout mutants for ACBP6 and other family members to verify specificity . For definitive specificity testing, conduct direct binding assays with multiple positive and negative controls, including isotype-matched irrelevant antibodies and chemically similar but antigenically unrelated compounds . If cross-reactivity persists, consider generating new antibodies against unique epitopes of ACBP6 with minimal homology to other ACBP family members.

What strategies work best for detecting low-abundance At5g10340/ACBP6 protein in plant samples?

For detecting low-abundance ACBP6 protein, optimize several aspects of the experimental protocol. Start with sample enrichment techniques such as immunoprecipitation or subcellular fractionation to concentrate the target protein. Increase protein loading amounts while ensuring the gel can resolve the small 10.4-kD ACBP6 protein effectively . Extend primary antibody incubation time (overnight at 4°C) and optimize antibody concentration through careful titration. Implement highly sensitive detection systems such as enhanced chemiluminescence (ECL) substrates or fluorescent secondary antibodies with digital imaging. Consider signal amplification methods like biotin-streptavidin systems or tyramide signal amplification for immunohistochemistry applications. For quantitative comparisons, use internal standards of known ACBP6 concentration. To enhance detection in tissues where ACBP6 is upregulated, such as plants exposed to cold stress, collect samples at timepoints of peak expression (e.g., 48 hours after 4°C treatment) .

How should researchers design experiments to study At5g10340/ACBP6 interactions with other proteins?

Studying ACBP6 protein interactions requires multiple complementary approaches. Begin with co-immunoprecipitation experiments using the ACBP6 antibody under mild, non-denaturing conditions to preserve protein-protein interactions. Pre-clear lysates with pre-immune serum or control IgG to remove non-specific binding proteins. Consider chemical crosslinking before cell lysis to capture transient interactions. Validate potential interaction partners through reciprocal co-IP, yeast two-hybrid assays, or in vitro binding studies. For demonstrating co-localization, combine ACBP6 antibody with antibodies against candidate interacting proteins in immunofluorescence microscopy. Implement proximity ligation assays for detecting interactions in situ with high sensitivity. Controls should include samples from acbp6 knockout plants and immunoprecipitations with non-specific antibodies of the same isotype. For comprehensive identification of the ACBP6 interactome, perform IP-mass spectrometry analysis followed by bioinformatic filtering to remove common contaminants and validation of top candidates through targeted approaches.

How should researchers quantify and normalize At5g10340/ACBP6 expression data from Western blot analyses?

Quantification of ACBP6 expression from Western blot analyses requires rigorous methodology. Begin by capturing digital images of blots within the linear range of detection, avoiding oversaturated signals. Use densitometry software to quantify band intensity for both ACBP6 and loading controls. For loading normalization, choose appropriate reference proteins that remain stable under your experimental conditions or use total protein staining (Ponceau S or similar). Calculate the ratio of ACBP6 signal to loading control for each sample, then express data as fold-change relative to control conditions. Include both technical replicates (multiple lanes of the same sample) and biological replicates (independent experiments) to assess variability. For time-course experiments of cold-induced expression changes, normalize all timepoints to their respective controls rather than a single baseline . Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions) to determine significance of observed changes. Present data graphically with error bars representing standard deviation or standard error, clearly indicating sample size and statistical significance.

How can researchers reconcile discrepancies between At5g10340/ACBP6 protein levels detected by antibody versus transcript levels measured by RT-qPCR?

Discrepancies between protein and transcript levels may arise from several biological and technical factors. First, verify antibody specificity and qPCR primer specificity to ensure both are detecting the intended targets. Consider temporal dynamics, as protein levels typically lag behind transcript levels due to the time required for translation. Examine protein stability and turnover rates, which may differ from mRNA degradation rates. Assess post-transcriptional regulation mechanisms including miRNA-mediated silencing or translational efficiency. For ACBP6, both northern-blot and western-blot analyses showed coordinated induction after cold treatment , but in cases of discrepancy, perform polysome profiling to assess translational status or pulse-chase experiments to determine protein half-life. Technical considerations include different extraction efficiencies for proteins versus RNA and differences in detection sensitivity between methods. When presenting discrepant results, clearly acknowledge the differences and propose biological mechanisms that might explain them rather than dismissing either dataset.

What controls are essential when comparing At5g10340/ACBP6 localization results from antibody-based methods versus fluorescent protein fusions?

When comparing ACBP6 localization results from different methodologies, implement comprehensive controls for valid interpretation. For antibody-based methods, include negative controls (acbp6 knockout tissues), specificity controls (peptide competition), and positive controls (tissues with known ACBP6 expression). For fluorescent protein fusions, use free fluorescent protein expression as a localization control and include multiple independent transgenic lines to rule out position effects . Confirm that fusion proteins remain functional through complementation tests in acbp6 mutant backgrounds. Cross-validate results between methods; previous research confirmed cytosolic localization of ACBP6 through both ACBP6-GFP fluorescence and antibody detection in subcellular fractions . Consider potential artifacts: antibody accessibility issues may affect immunolocalization, while fluorescent tags might disrupt localization signals. Test both N- and C-terminal fusions to rule out tag interference. For quantitative comparisons between methods, standardize image acquisition parameters and analysis protocols. When discrepancies arise, consider biological explanations (developmental stage, stress conditions) before assuming methodological artifacts.

How can researchers adapt At5g10340/ACBP6 antibody protocols for super-resolution microscopy applications?

Adapting ACBP6 antibody protocols for super-resolution microscopy requires optimization of several parameters. Start with fixation method selection, comparing paraformaldehyde, glutaraldehyde, or methanol to determine which best preserves antigenicity while maintaining cellular architecture. Optimize antibody concentration through careful titration experiments, typically using higher dilutions than conventional immunofluorescence to reduce background. Select secondary antibodies conjugated to bright, photostable fluorophores specifically designed for super-resolution techniques (e.g., Alexa Fluor 647 for STORM). For multi-color imaging, ensure minimal spectral overlap between fluorophores. Implement rigorous controls including acbp6 knockout samples and peptide competition assays. Consider direct labeling of primary antibodies to reduce the linkage error introduced by secondary antibodies. For correlative approaches, combine ACBP6 antibody labeling with ACBP6-GFP expression to validate findings across methods . The cytosolic localization of ACBP6 makes it particularly suitable for super-resolution techniques that excel at resolving diffuse protein distributions within distinct cellular compartments.

What experimental design considerations are important when using At5g10340/ACBP6 antibody to study protein modifications during plant stress responses?

When studying ACBP6 modifications during stress responses, implement a comprehensive experimental design addressing several key considerations. First, establish a detailed time-course sampling strategy that captures both early responses and sustained changes, as ACBP6 shows increased expression 48 hours after cold treatment . Include parallel analysis of both total ACBP6 levels and specific modifications using modification-specific antibodies when available. For post-translational modifications like phosphorylation, consider phosphatase inhibitors during extraction and separation techniques like Phos-tag gels that can resolve modified forms. Implement two-dimensional gel electrophoresis followed by Western blotting to separate modified ACBP6 isoforms. For definitive identification of modifications, combine immunoprecipitation using the ACBP6 antibody with mass spectrometry analysis. Essential controls include unmodified recombinant ACBP6 protein, enzymatic removal of specific modifications (e.g., phosphatase treatment), and samples from acbp6 mutants. Compare modifications across different stress conditions (cold, drought, salt) to identify stress-specific versus general stress response patterns.

How can researchers validate contradictory results when studying At5g10340/ACBP6 protein-lipid interactions using different methodological approaches?

Validating contradictory results in ACBP6 protein-lipid interaction studies requires systematic investigation through multiple methodologies. First, carefully evaluate the experimental conditions of each approach, considering differences in pH, temperature, ion concentration, and redox environment that might affect interactions. Implement both in vitro approaches using purified components and in vivo approaches within the cellular context. For in vitro validation, perform lipid-binding assays using recombinant ACBP6 with defined lipid species under standardized conditions. Use biophysical methods such as isothermal titration calorimetry, surface plasmon resonance, or microscale thermophoresis to determine binding parameters (Kd, stoichiometry). For in vivo validation, combine co-localization studies using the ACBP6 antibody with lipid probes, pull-down experiments using lipid-conjugated beads, and proximity labeling approaches. Include structure-function analysis by examining mutations in predicted lipid-binding domains of ACBP6. The cytosolic localization of ACBP6 confirmed by antibody detection in subcellular fractions provides important context for interpreting lipid interaction data . When presenting contradictory results, clearly delineate methodological differences and propose models that integrate findings rather than dismissing particular approaches.