ACS1 Antibody

What is ACS1 Antibody and what biological systems is it used to study?

ACS1 antibody is an immunological reagent designed to recognize and bind specifically to 1-aminocyclopropane-1-carboxylate synthase-like protein 1. This protein is part of the ACC synthase family (EC 4.4.1.14), which catalyzes the conversion of S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene. ACS1 is particularly noteworthy as it is transcriptionally active but enzymatically inactive, suggesting potential regulatory functions beyond direct catalytic activity .

The antibody is primarily used in plant science research, particularly in Arabidopsis thaliana studies, where the gene is also known as AT-ACS1 or ARABIDOPSIS THALIANA 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 1. Research applications include investigating ethylene signaling pathways, plant stress responses, and developmental processes regulated by ethylene.

What are the proper storage and handling recommendations for ACS1 Antibody?

Optimal storage and handling of ACS1 antibody is critical for maintaining its functionality and specificity. The antibody is typically supplied in lyophilized form and requires specific handling protocols:

Improper storage can lead to antibody degradation, resulting in decreased sensitivity and increased background in experimental applications .

How can researchers validate the specificity of ACS1 Antibody?

Validating antibody specificity is a critical step that ensures experimental results accurately reflect ACS1 biology. Multiple approaches should be employed:

• Western blot analysis with positive and negative controls: Include wild-type tissue samples alongside ACS1 knockout/knockdown samples to verify specific binding patterns.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to verify that binding is eliminated in the presence of the specific antigen.

• Cross-reactivity testing: Test the antibody against related ACC synthase family members (ACS2-ACS12) to ensure specificity for ACS1.

• Immunoprecipitation followed by mass spectrometry: This approach provides definitive identification of the captured proteins and can reveal potential cross-reactivity.

• Multiple antibody verification: When possible, use antibodies targeting different epitopes of ACS1 to confirm results across methodologies.

These validation approaches should be documented systematically to establish confidence in experimental findings when using the ACS1 antibody.

How can researchers differentiate between transcriptional activity and enzymatic inactivity of ACS1 using antibody-based techniques?

Since ACS1 is transcriptionally active but enzymatically inactive , distinguishing between these properties requires complementary experimental approaches:

• Combined immunoprecipitation and activity assays: Immunoprecipitate ACS1 using the specific antibody, then subject the precipitate to enzymatic activity assays measuring ACC production from SAM. Compare activity to other ACS family members.

• Proximity labeling combined with antibody detection: Use techniques like BioID or APEX2 fused to ACS1 to identify interacting proteins, then confirm interactions using co-immunoprecipitation with the ACS1 antibody.

• Chromatin immunoprecipitation (ChIP) analysis: If ACS1 has regulatory functions, ChIP using ACS1 antibody can identify potential DNA binding sites or associations with transcriptional complexes.

• Structural analysis of epitope accessibility: Compare epitope exposure between active and inactive conformational states using techniques like hydrogen-deuterium exchange mass spectrometry combined with antibody binding analysis.

These approaches can help determine whether the inactive enzyme status is due to structural constraints, post-translational modifications, or other regulatory mechanisms.

What experimental approaches can identify post-translational modifications of ACS1 using antibody-based methods?

Post-translational modifications (PTMs) often regulate protein function and may explain ACS1's enzymatic inactivity. Researchers can employ these approaches:

When analyzing potential phosphorylation sites, researchers should consider using techniques similar to those employed in antibody array systems that can capture and analyze multiple modified forms simultaneously .

How can advanced computational modeling enhance ACS1 antibody epitope mapping and binding prediction?

Computational approaches can significantly improve understanding of ACS1 antibody interactions:

• Structural epitope prediction: Using techniques similar to those in generative AI antibody design , researchers can predict the conformational epitopes recognized by ACS1 antibodies.

• Molecular dynamics simulations: Simulate antibody-antigen binding to predict affinity and specificity, particularly useful when designing experiments with limited antibody availability.

• Paratope-epitope interaction mapping: Similar to approaches used in HIV-1 antibody studies , map the specific amino acid interactions between antibody CDRs and ACS1 epitopes.

• Machine learning models for cross-reactivity prediction: Train models on antibody binding data to predict potential cross-reactivity with other ACC synthase family members.

• In silico mutagenesis analysis: Predict how mutations in ACS1 might affect antibody binding, guiding experimental design for specificity testing.

These computational approaches can guide experimental design and help interpret results, particularly when combined with experimental validation through techniques like surface plasmon resonance or bio-layer interferometry.

What are the optimal protocols for immunoprecipitation of ACS1 from plant tissues?

Effective immunoprecipitation of ACS1 requires careful optimization:

• Tissue preparation and lysis:

  • Harvest plant tissues quickly and flash-freeze in liquid nitrogen

  • Grind tissues to fine powder under liquid nitrogen

  • Extract in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors

  • Clear lysate by centrifugation (16,000 × g, 10 min, 4°C)

• Antibody binding:

  • Pre-clear lysate with protein A/G beads (1 hour, 4°C)

  • Incubate cleared lysate with ACS1 antibody (2-5 μg per mg of total protein) overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads and incubate for 2-3 hours at 4°C

• Washing and elution:

  • Wash beads 4-5 times with wash buffer (lysis buffer with reduced detergent)

  • Elute bound proteins with SDS sample buffer or low pH glycine buffer

  • Analyze by western blotting using the same or different ACS1 antibody

• Controls:

  • Include IgG control from the same species as the ACS1 antibody

  • Include samples from ACS1 knockout/knockdown plants as negative controls

This protocol can be adapted for co-immunoprecipitation studies to identify ACS1 interaction partners, providing insights into its non-enzymatic functions.

How can researchers optimize western blot protocols specifically for ACS1 detection?

Optimizing western blot protocols for ACS1 detection requires attention to several key parameters:

• Sample preparation:

  • Include reducing agents (DTT or β-mercaptoethanol) in sample buffer

  • Heat samples at 95°C for 5 minutes to ensure complete denaturation

  • Load adequate protein amounts (typically 20-50 μg per lane)

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of ACS1 protein

  • Include molecular weight markers to verify ACS1 band size

  • Run at constant voltage (100-120V) for consistent results

• Transfer conditions:

  • Use PVDF membranes for better protein retention and antibody binding

  • Transfer at 100V for 1 hour in cold transfer buffer with 20% methanol

  • Verify transfer efficiency using reversible staining (Ponceau S)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBS-T for 1 hour at room temperature

  • Incubate with ACS1 antibody at optimized dilution (typically 1:1000) overnight at 4°C

  • Wash thoroughly (4 × 5 minutes) with TBS-T before secondary antibody

• Detection optimization:

  • Use enhanced chemiluminescence detection for standard applications

  • Consider fluorescent secondary antibodies for multiplex detection and quantification

  • Optimize exposure times to prevent signal saturation

• Troubleshooting:

  • For high background, increase washing steps and optimize antibody dilution

  • For weak signals, increase antibody concentration or extend incubation time

  • For non-specific bands, use additional blocking agents (BSA or casein)

This optimized protocol should be validated across different plant tissue types and growth conditions.

What are the best approaches for using ACS1 antibody in immunohistochemistry and immunofluorescence studies?

Successful immunohistochemistry and immunofluorescence with ACS1 antibody requires careful tissue preparation and staining protocols:

• Tissue fixation and processing:

  • Fix plant tissues in 4% paraformaldehyde for 12-24 hours at 4°C

  • Dehydrate through ethanol series (70%, 80%, 90%, 100%)

  • Embed in paraffin or optimal cutting temperature (OCT) compound

  • Section at 5-10 μm thickness for optimal antibody penetration

• Antigen retrieval:

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Alternatively, use enzymatic retrieval with proteinase K for certain applications

  • Optimize retrieval conditions to balance antigen exposure and tissue preservation

• Antibody staining:

  • Block with 5% normal serum from the same species as the secondary antibody

  • Incubate with ACS1 antibody at optimized dilution (1:50 to 1:200) overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies for immunofluorescence

  • Include DAPI or other nuclear counterstains for cellular context

• Controls and validation:

  • Include sections without primary antibody to assess background

  • Use tissues from ACS1 knockout plants as negative controls

  • Consider dual staining with antibodies to known cellular markers

• Imaging considerations:

  • Use confocal microscopy for subcellular localization studies

  • Apply deconvolution algorithms to enhance resolution

  • Perform z-stack imaging to capture 3D distribution

These protocols should be adapted based on the specific plant tissue being examined and the research question being addressed.

How can ACS1 antibody be incorporated into antibody microarray platforms for high-throughput studies?

Integrating ACS1 antibody into microarray platforms enables high-throughput analysis similar to approaches used in clinical research settings :

• Array preparation:

  • Spot ACS1 antibody onto nitrocellulose-coated glass slides using automated systems

  • Use microarraying robots to ensure consistent spot size and spacing

  • Include graduated concentrations for quantitative analysis

  • Add control antibodies (isotype controls, positive controls) to validate results

• Sample preparation:

  • Extract proteins from plant tissues using optimized buffers

  • Label extracted proteins with fluorescent dyes (Cy3, Cy5)

  • Apply labeled samples to arrays and incubate under optimized conditions

• Data acquisition and analysis:

  • Scan arrays using high-resolution fluorescence scanners

  • Normalize signal intensities to control spots

  • Apply statistical analysis to identify significant differences

  • Validate key findings with orthogonal methods (western blot, ELISA)

• Multiplexed analysis:

  • Design arrays containing antibodies against multiple proteins in ethylene signaling

  • Include antibodies targeting different epitopes of ACS1

  • Add antibodies against known ACS1 interaction partners

This technology enables simultaneous assessment of ACS1 expression across multiple experimental conditions or genetic backgrounds.

What are the considerations for using ACS1 antibody in CRISPR-engineered plant systems?

When applying ACS1 antibody in CRISPR-engineered systems, researchers should consider:

• Epitope preservation verification:

  • Ensure CRISPR edits don't alter the epitope recognized by the antibody

  • Design sgRNAs to target regions away from antibody binding sites

  • Validate antibody binding to edited protein by western blot

• Tagging strategies:

  • Consider CRISPR knock-in of epitope tags (FLAG, HA, V5) for enhanced detection

  • Use the ACS1 antibody alongside tag-specific antibodies to confirm specificity

  • Verify that tags don't interfere with protein function or localization

• Quantifying editing efficiency:

  • Use ACS1 antibody in flow cytometry or cell sorting applications

  • Analyze mixed populations of edited and non-edited cells

  • Correlate protein levels with genomic editing efficiency

• Analyzing protein-protein interactions:

  • Apply techniques similar to zero-shot antibody design approaches to predict how CRISPR modifications might affect binding

  • Use ACS1 antibody in proximity ligation assays to detect novel interactions

  • Compare interaction networks between wild-type and edited ACS1 variants

• Functional validation:

  • Use antibody to verify expression of CRISPR-edited ACS1 variants

  • Combine with activity assays to correlate structural changes with function

  • Perform immunoprecipitation followed by mass spectrometry to identify post-translational modifications in edited variants

These considerations ensure reliable detection and characterization of ACS1 in genome-edited plant systems, enabling more sophisticated functional studies.

How can electron microscopy be combined with ACS1 antibody for ultrastructural studies?

Combining electron microscopy with immunolabeling techniques offers powerful insights into ACS1 localization at the ultrastructural level:

• Sample preparation for immunoelectron microscopy:

  • Fix plant tissues in 2% glutaraldehyde in sodium cacodylate buffer

  • Post-fix with 1% osmium tetroxide

  • Dehydrate through ethanol series and embed in appropriate resin

  • Section at ultrathin levels (70-90 nm) for transmission electron microscopy

• Immunogold labeling protocol:

  • Etch sections with sodium metaperiodate to expose epitopes

  • Block with normal serum or BSA to reduce non-specific binding

  • Incubate with ACS1 antibody at optimized dilution (typically 1:10 to 1:50)

  • Apply gold-conjugated secondary antibodies (typically 10-15 nm gold particles)

  • Counterstain with uranyl acetate and lead citrate

• Controls and validation:

  • Include sections without primary antibody

  • Use pre-immune serum as negative control

  • Apply statistical analysis to quantify gold particle distribution

• Alternative approaches:

  • Consider scanning electron microscopy for surface analysis following protocols similar to those described in search result

  • Use correlative light and electron microscopy (CLEM) to bridge fluorescence and ultrastructural data

  • Apply cryo-electron microscopy techniques similar to those used in antibody-antigen structural studies

These approaches can reveal the precise subcellular localization of ACS1 and its potential association with specific organelles or membrane structures, providing insights into its non-enzymatic functions.

What are the emerging technologies that will enhance ACS1 antibody applications in research?

Several cutting-edge technologies are poised to revolutionize ACS1 antibody applications:

• AI-driven antibody engineering: Similar to recent advances in zero-shot antibody design , computational approaches may soon enable the development of ACS1 antibodies with enhanced specificity and sensitivity.

• Single-cell proteomics: Emerging techniques will allow detection of ACS1 at the single-cell level, revealing cell-type specific expression patterns in complex plant tissues.

• Spatial transcriptomics integrated with antibody detection: Combining spatial transcriptomics with antibody-based protein detection will correlate ACS1 transcription and translation in intact tissues.

• Nanobody development: Smaller antibody fragments derived from camelid antibodies offer improved tissue penetration and potentially higher resolution imaging.

• Multiplex imaging technologies: Techniques like imaging mass cytometry and CODEX will enable simultaneous detection of ACS1 alongside dozens of other proteins in the same tissue section.

• Proximity labeling proteomics: Methods like BioID and APEX2 will provide comprehensive interaction maps for ACS1 when combined with specific antibodies.

These technological advances will significantly expand our understanding of ACS1's role in plant biology and ethylene signaling pathways, potentially revealing novel functions beyond its known transcriptionally active but enzymatically inactive status .