At1g52360 Antibody

What is At1g52360 protein and what is its biological significance?

At1g52360 encodes β1-COP, a critical component of the Coat Protein Complexes (COPs) in Arabidopsis thaliana. The protein plays an essential role in vesicular transport, particularly in endomembrane trafficking systems. Most significantly, research has demonstrated that both β1-COP and β2-COP are required for proper female and male gametophyte development in plants . This makes the protein an important target for researchers studying plant reproductive biology and membrane trafficking.

The protein is documented in the UniProt database under accession number Q9C827 and is encoded by a protein-coding gene located on chromosome 1 of the Arabidopsis genome . Understanding this protein's function provides critical insights into fundamental plant cellular processes.

What applications is the At1g52360 antibody validated for?

The At1g52360 antibody has been specifically tested and validated for the following research applications:

Both applications ensure proper identification of the target antigen and can be optimized for specific experimental conditions . For researchers planning to use this antibody in other applications such as immunohistochemistry or immunoprecipitation, preliminary validation experiments would be necessary as these applications are not currently listed among tested applications.

What are the optimal storage and handling conditions for At1g52360 antibody?

To maintain antibody integrity and activity, the At1g52360 antibody should be stored at either -20°C or -80°C immediately upon receipt . The storage buffer contains 50% glycerol and 0.01M PBS at pH 7.4 with 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during freeze-thaw cycles.

Best practices for handling include:

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

• Aliquot the antibody into smaller volumes upon receipt

• Thaw aliquots completely before use and mix gently by inversion

• Keep on ice when in use during experiments

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

Following these storage guidelines will help ensure consistent experimental results and maximize the usable lifetime of the antibody.

What is the recommended Western blot protocol for At1g52360 antibody?

When using At1g52360 antibody for Western blot analysis, researchers should follow this optimized protocol:

• Sample Preparation:

  • Extract total protein from Arabidopsis thaliana tissues using a plant protein extraction buffer containing protease inhibitors

  • Quantify protein concentration using Bradford or BCA assay

  • Prepare 20-50 μg of total protein per lane

• Gel Electrophoresis:

  • Separate proteins on a 10-12% SDS-PAGE gel

  • Include molecular weight markers to confirm target protein size

• Transfer:

  • Transfer proteins to a PVDF or nitrocellulose membrane using standard wet or semi-dry transfer methods

  • Confirm transfer efficiency with Ponceau S staining

• Blocking:

  • Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary Antibody Incubation:

  • Dilute At1g52360 antibody 1:1000 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash membrane 3-5 times with TBST, 5 minutes per wash

• Secondary Antibody Incubation:

  • Use anti-rabbit IgG secondary antibody conjugated to HRP at 1:5000 dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Visualize using ECL substrate and appropriate imaging system

Including appropriate negative and positive controls is essential for proper interpretation of results. The antibody should detect a protein of the expected molecular weight for At1g52360.

How can researchers optimize ELISA protocols with At1g52360 antibody?

For optimal ELISA performance with At1g52360 antibody, consider the following protocol recommendations:

• Plate Coating:

  • Coat 96-well plate with capture antibody or antigen at 1-10 μg/ml in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

• Blocking:

  • Block with 2-5% BSA in PBS for 1-2 hours at room temperature

• Sample Preparation:

  • Prepare standard curve using recombinant At1g52360 protein

  • Process plant samples with appropriate extraction buffer

  • Perform serial dilutions to ensure readings fall within the linear range

• Antibody Dilution Optimization:

  • Perform checkerboard titration to determine optimal concentration

  • Typical starting dilution range: 1:500 to 1:5000

• Incubation Conditions:

  • Primary antibody: 1-2 hours at room temperature or overnight at 4°C

  • Secondary antibody: 1 hour at room temperature

• Signal Development:

  • Use TMB substrate for HRP-conjugated secondary antibodies

  • Monitor color development and stop reaction with 2N H₂SO₄ when appropriate

• Data Analysis:

  • Generate standard curve and calculate sample concentrations

For quantitative analysis, a standard curve using purified recombinant At1g52360 protein is recommended. This approach will enable precise measurement of protein levels across different samples.

What controls should be included when performing experiments with At1g52360 antibody?

Proper experimental controls are crucial for generating reliable and interpretable results with At1g52360 antibody:

Including these controls will significantly enhance data quality and reliability. The pre-immune serum and antigen controls are particularly valuable as they are specifically provided with the antibody for validation purposes .

How can At1g52360 antibody be utilized to study plant gametophyte development?

Given that β1-COP (encoded by At1g52360) is essential for gametophyte development in Arabidopsis , the antibody can be employed in multiple advanced research approaches:

• Immunolocalization Studies:

  • Perform immunofluorescence on developing anthers and ovules

  • Use confocal microscopy to track β1-COP localization during different stages of gametophyte development

  • Co-localize with other endomembrane markers to understand trafficking dynamics

• Developmental Time Course Analysis:

  • Extract proteins from flowers at different developmental stages

  • Perform quantitative Western blots to measure β1-COP expression levels

  • Correlate protein levels with specific developmental events

• Co-immunoprecipitation (Co-IP):

  • Use At1g52360 antibody for pull-down experiments

  • Identify interaction partners specifically in reproductive tissues

  • Compare interactome differences between vegetative and reproductive tissues

• ChIP-Seq Studies:

  • Perform chromatin immunoprecipitation if studying transcription factors that regulate At1g52360

  • Map binding sites and regulatory elements controlling expression during gametogenesis

These approaches can provide valuable insights into the molecular mechanisms by which coat protein complexes influence gametophyte development, potentially identifying novel therapeutic targets or genetic engineering strategies for crop improvement.

What techniques can reveal At1g52360 interactions with other Coat Protein Complex components?

To investigate the interaction network of At1g52360 (β1-COP) with other components of Coat Protein Complexes, researchers can employ these advanced techniques:

• Proximity-dependent Biotin Identification (BioID):

  • Generate fusion constructs of At1g52360 with a biotin ligase

  • Express in Arabidopsis to biotinylate proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

• Förster Resonance Energy Transfer (FRET):

  • Create fluorescent protein fusions with At1g52360 and suspected interaction partners

  • Measure energy transfer to quantify protein-protein interactions in vivo

  • Track interactions in real-time during vesicle formation

• Bimolecular Fluorescence Complementation (BiFC):

  • Split YFP or other fluorescent protein into two non-fluorescent fragments

  • Fuse fragments to At1g52360 and potential interaction partners

  • Observe fluorescence restoration when proteins interact

• Cryo-electron Microscopy:

  • Isolate intact coat protein complexes containing At1g52360

  • Determine structural organization at near-atomic resolution

  • Map interaction interfaces between β1-COP and other components

• Yeast Three-hybrid System:

  • Investigate potential RNA-mediated interactions between protein components

  • Particularly useful if regulatory RNAs influence complex assembly

These approaches provide complementary information about the composition, dynamics, and structural organization of coat protein complexes containing At1g52360, contributing to a comprehensive understanding of vesicular transport mechanisms in plants.

How can researchers troubleshoot non-specific binding when using At1g52360 antibody?

When encountering non-specific binding issues with At1g52360 antibody, researchers should implement the following troubleshooting strategies:

• Optimize Antibody Concentration:

  • Perform titration experiments (1:500 to 1:5000 dilutions)

  • Use the highest dilution that gives specific signal while minimizing background

• Modify Blocking Conditions:

  • Test different blocking agents (milk vs. BSA vs. serum)

  • Increase blocking time or agent concentration

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Adjust Washing Protocol:

  • Increase number and duration of wash steps

  • Use higher stringency wash buffers (increased salt concentration)

• Pre-adsorb Antibody:

  • Incubate diluted antibody with protein extract from knockout/knockdown plants

  • Remove antibodies that bind to non-specific targets

• Validate with Alternative Detection Methods:

  • Compare results with alternative detection methods like mass spectrometry

  • Use additional antibodies targeting different epitopes of the same protein

• Epitope Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific bands should disappear while non-specific bands remain

• Sample Preparation Modifications:

  • Optimize protein extraction method to reduce interfering compounds

  • Include additional protease inhibitors to prevent degradation products

The polyclonal nature of the At1g52360 antibody means it recognizes multiple epitopes, which can increase sensitivity but may also contribute to cross-reactivity . Using the pre-immune serum provided as a negative control is particularly valuable for distinguishing specific from non-specific signals .

What advanced imaging techniques can be combined with At1g52360 antibody for subcellular localization studies?

For detailed subcellular localization of At1g52360 (β1-COP), researchers can combine the antibody with these sophisticated imaging approaches:

• Super-resolution Microscopy:

  • Techniques: STED, PALM, STORM

  • Resolution: Can achieve 20-50 nm resolution compared to ~200 nm in conventional microscopy

  • Application: Visualize individual vesicles and precise co-localization with other coat proteins

• Correlative Light and Electron Microscopy (CLEM):

  • Process: Combine immunofluorescence with transmission electron microscopy

  • Benefit: Link protein localization to ultrastructural context

  • Implementation: Use gold-conjugated secondary antibodies for EM detection

• Live Cell Imaging with Complementary Probes:

  • Approach: Use At1g52360 antibody in fixed cells alongside live-cell imaging of fluorescent-tagged proteins

  • Markers: Combine with established endomembrane markers (Golgi, ER, TGN)

  • Analysis: Track dynamic processes through time-lapse imaging

• Expansion Microscopy:

  • Technique: Physically expand specimens using hydrogel embedding

  • Advantage: Achieves super-resolution using standard confocal microscopes

  • Application: Resolve closely positioned coat proteins in complex structures

• FRAP/FLIP Analysis with Immunolocalization:

  • Method: Combine photobleaching recovery studies of fluorescent proteins with antibody localization

  • Output: Correlate protein dynamics with steady-state distribution

  • Insight: Determine mobile and immobile fractions of coat proteins

These advanced imaging approaches, when combined with the specificity of At1g52360 antibody, can provide unprecedented insights into the dynamic behavior and precise localization of coat protein complexes during vesicle formation and transport in plant cells.

How should experiments be designed to study At1g52360 expression across plant developmental stages?

To effectively investigate At1g52360 expression patterns throughout plant development, researchers should consider this comprehensive experimental design:

• Tissue Sampling Strategy:

  • Collect multiple tissue types (roots, leaves, stems, flowers, siliques)

  • Sample at defined developmental stages using standardized growth conditions

  • Include reproductive tissues at various developmental points

• Quantitative Analysis Methods:

  • Western blot with densitometry for protein quantification

  • qRT-PCR for transcript level analysis

  • Correlate protein and transcript data for post-transcriptional regulation insights

• Standardization Approaches:

  • Use consistent protein extraction methods across all samples

  • Include internal reference proteins (actin, tubulin) for normalization

  • Process biological replicates (minimum n=3) for statistical validity

• Data Presentation:

  • Express results as fold change relative to a reference tissue

  • Include statistical analysis (ANOVA with post-hoc tests)

  • Present data in heat maps to visualize expression patterns

This systematic approach will provide robust data on At1g52360 expression dynamics throughout plant development, potentially revealing tissue-specific functions beyond the known roles in gametophyte development .

What methodological considerations are important when studying At1g52360 under stress conditions?

When investigating At1g52360 responses to environmental stresses, researchers should implement these methodological considerations:

• Stress Application Protocols:

  • Define precise stress parameters (duration, intensity)

  • Apply stresses at consistent developmental stages

  • Include recovery periods to assess response dynamics

• Stress Types to Consider:

  Stress Category	Specific Conditions	Relevance
  Abiotic	Drought, salt, temperature extremes, heavy metals	May affect vesicular trafficking
  Biotic	Pathogens, herbivory, symbiotic interactions	Could alter secretory pathway activity
  Hormone	ABA, ethylene, jasmonic acid treatment	Known to modify endomembrane dynamics

• Multi-level Analysis Approach:

  • Transcriptional: qRT-PCR to assess mRNA level changes

  • Translational: Polysome profiling to detect translation efficiency

  • Post-translational: Western blot to measure protein abundance

  • Functional: Vesicle trafficking assays to assess activity

• Controls and References:

  • Include untreated controls for each time point

  • Use known stress-responsive genes as positive controls

  • Compare responses to related genes (e.g., β2-COP)

This comprehensive approach will reveal whether At1g52360 expression and function are modulated during stress responses, potentially uncovering novel roles beyond its characterized function in gametophyte development .

What are the current knowledge gaps regarding At1g52360 function and future research directions?

Despite our understanding of At1g52360 as a component of coat protein complexes essential for gametophyte development , several significant knowledge gaps remain:

• Mechanistic Understanding:

  • Precise molecular mechanism by which β1-COP influences gametogenesis

  • Temporal and spatial dynamics of At1g52360 during reproductive development

  • Functional redundancy between β1-COP and β2-COP in different tissues

• Interactome Characterization:

  • Complete interaction network of At1g52360 in different cell types

  • Regulatory factors controlling At1g52360 expression and function

  • Post-translational modifications affecting protein activity

• Evolutionary Conservation:

  • Functional conservation of At1g52360 across plant species

  • Specialized roles in crop plants versus model organisms

  • Adaptive changes in coat protein complexes during plant evolution

Future research directions should focus on combining genetic approaches (CRISPR/Cas9 genome editing, conditional knockouts) with advanced imaging and biochemical techniques using At1g52360 antibody. Particular attention should be given to tissue-specific functions and potential roles in plant stress responses, as membrane trafficking pathways are often reconfigured during adaptation to environmental challenges.