At5g52605 Antibody

What is At5g52605 and why is it important in plant research?

At5g52605 encodes a putative defensin-like protein 20 in Arabidopsis thaliana, consisting of 90 amino acids with the sequence: MVRTNVVSFVLFAAIVLCILYGQKHIAPWIFEDKSICCKEHPSVGRCLPGIDDDAEKDGKCWKFCIEGCETGGFCKLFEHKHICHCNCSG . Defensin-like proteins are small cysteine-rich peptides that play critical roles in plant immunity against pathogens, abiotic stress responses, and developmental processes. The study of At5g52605 contributes to our understanding of plant defense mechanisms and potentially informs agricultural applications for enhancing crop resistance.

When designing experiments to investigate At5g52605 function, researchers should consider:

• Expression patterns across different tissues and developmental stages

• Response to various biotic and abiotic stresses

• Subcellular localization

• Potential interaction partners

• Phenotypic effects of gene knockout or overexpression

What types of At5g52605 antibodies are available for research?

Several monoclonal antibody combinations are available for detecting At5g52605 protein in experimental settings, targeting different regions of the protein:

Each antibody combination has an ELISA titer of approximately 10,000, corresponding to detection sensitivity of about 1 ng of target protein in Western blot applications . These antibodies are generated against synthetic peptide antigens representing different regions of the At5g52605 protein.

How should I design experiments using At5g52605 antibodies?

Effective experimental design for At5g52605 antibody research requires careful consideration of variables, controls, and proper methodology:

• Define your variables clearly:

  • Independent variable: Treatment conditions affecting At5g52605 expression

  • Dependent variable: Measurable outcomes (protein levels, localization patterns)

  • Control variables: Growth conditions, tissue types, developmental stages

• Include appropriate controls:

  • Positive control: Known At5g52605-expressing tissues

  • Negative control: Tissues where At5g52605 is not expressed or knockout mutants

  • Loading controls: Housekeeping proteins to normalize protein amounts

• Optimize antibody conditions:

  • Determine appropriate antibody dilution through titration experiments

  • Test blocking conditions to minimize non-specific binding

  • Validate antibody specificity using recombinant protein or knockout lines

• Select appropriate detection methods:

  • Western blot for protein expression levels

  • Immunohistochemistry for localization studies

  • ELISA for quantitative measurements

  • Immunoprecipitation for protein interaction studies

Following these experimental design principles will help ensure reliable and reproducible results when using At5g52605 antibodies in research settings.

How can I validate the specificity of At5g52605 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For At5g52605 antibodies, consider the following validation approaches:

• Western blot analysis:

  • Compare wild-type and At5g52605 knockout/knockdown plants

  • Verify single band of expected molecular weight (approximately 10 kDa for mature protein)

  • Pre-absorb antibody with recombinant At5g52605 protein to confirm specificity

• Epitope mapping:

  • Determine precise binding sites using epitope determination services ($100 per combination)

  • Map epitopes to verify they don't overlap with highly conserved regions in related proteins

• Cross-reactivity testing:

  • Test antibodies against closely related defensin-like proteins

  • Evaluate potential cross-reactivity with other cysteine-rich proteins

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm identity of pulled-down proteins

• Immunohistochemistry patterns:

  • Compare antibody staining patterns with mRNA expression data

  • Verify absence of signal in knockout tissues

A systematic validation approach using multiple methods provides confidence in antibody specificity and ensures experimental rigor.

What are common troubleshooting issues with At5g52605 antibody experiments?

When working with At5g52605 antibodies, researchers may encounter several technical challenges:

For plant-specific samples, consider that defensin-like proteins may be difficult to extract due to their small size and potential binding to cell walls. Using specialized extraction protocols with chaotropic agents can improve recovery of these proteins from plant tissues.

How can At5g52605 antibodies be utilized in antibody-cell conjugation (ACC) techniques?

Antibody-cell conjugation (ACC) represents an innovative approach for targeted cell therapy that can be adapted for plant research applications. For At5g52605 studies, researchers can apply ACC principles to create targeted delivery systems:

• Chemical conjugation methods:

  • NHS-DNA coupling can be used to directly modify cell surfaces with At5g52605 antibodies

  • This approach involves a three-step process: coupling single-stranded DNA to the antibody, coupling complementary DNA to cell surface proteins, and joining through DNA hybridization

• Metabolic sugar engineering approach:

  • Introduce azide moieties onto plant cell surfaces through metabolic incorporation

  • Modify At5g52605 antibodies with DBCO-PEG4-NHS ester

  • Couple through bioorthogonal azide-alkyne click chemistry

• Chemoenzymatic coupling:

  • Use enzymatic methods such as transglutaminase or sortase to conjugate At5g52605 antibodies to plant cell surfaces

  • These methods offer site-specific conjugation with minimal interference to endogenous functions

These ACC techniques can be applied to create At5g52605 antibody-conjugated plant protoplasts or suspension cultures for studying protein function, cellular interactions, or developing novel biosensors for plant defense responses. The primary advantage of ACC over genetic engineering approaches is the rapid and controllable preparation time without genetic modification requirements .

What advanced microscopy techniques can be combined with At5g52605 antibodies?

Advanced microscopy approaches can provide deeper insights into At5g52605 localization, dynamics, and interactions:

• Super-resolution microscopy:

  • STORM (Stochastic Optical Reconstruction Microscopy): Achieves 20-30 nm resolution to precisely localize At5g52605 in subcellular compartments

  • SIM (Structured Illumination Microscopy): Provides detailed visualization of At5g52605 distribution in plant cells

  • Requires careful optimization of At5g52605 antibodies with appropriate fluorophores that support super-resolution imaging

• Multi-modal imaging:

  • Combine immunofluorescence with other imaging modalities like electron microscopy

  • Correlative Light and Electron Microscopy (CLEM): Localize At5g52605 at ultrastructural level

  • Protocol modification: Use gold-conjugated secondary antibodies for EM detection

• Live-cell imaging approaches:

  • Antibody fragment conjugation to cell-permeable peptides

  • Track dynamic changes in At5g52605 localization during stress responses

  • Consider using nanobody formats for improved penetration into live plant tissues

• Multiplexed imaging:

  • Simultaneously detect At5g52605 and interaction partners

  • Employ spectral unmixing to distinguish multiple fluorophores

  • Use sequential staining protocols to prevent antibody cross-reactivity

For plant cell applications, cell wall penetration represents a significant challenge. Pre-treatment with cell wall digesting enzymes or microinjection techniques may be necessary for successful immunolabeling with At5g52605 antibodies in intact plant tissues.

How can At5g52605 antibodies be employed in protein interaction studies?

Understanding protein-protein interactions is crucial for elucidating At5g52605 function in plant defense mechanisms:

• Co-immunoprecipitation (Co-IP):

  • Use X-Q2V2S1 antibodies to pull down At5g52605 and identify interacting partners

  • Optimization steps:

    • Cross-link proteins in vivo before extraction

    • Use plant-specific extraction buffers to maintain native interactions

    • Verify results with reciprocal Co-IP using antibodies against putative interactors

• Proximity ligation assay (PLA):

  • Detect in situ protein interactions with spatial resolution

  • Requires two antibodies recognizing different proteins (At5g52605 and candidate interactor)

  • Each antibody is conjugated to complementary oligonucleotides

  • Signal amplification occurs only when proteins are in close proximity (<40 nm)

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Confirm antibody-detected interactions using split fluorescent protein constructs

  • Compare BiFC results with Co-IP findings to validate interactions

• Antibody-based protein arrays:

  • Immobilize candidate interacting proteins on arrays

  • Probe with purified At5g52605 and detect with specific antibodies

  • Create comprehensive interaction maps for defensin-like protein networks

When studying plant defensin-like proteins, consider that interactions may be condition-dependent, occurring only during specific stress responses or developmental stages.

How can quantitative proteomics be integrated with At5g52605 antibody studies?

Integrating At5g52605 antibody techniques with quantitative proteomics provides comprehensive insights into protein function and regulation:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use X-Q2V2S1 antibodies to enrich At5g52605 and associated proteins

  • Analyze by LC-MS/MS to identify and quantify interaction partners

  • Compare different stress conditions to identify context-dependent interactions

  • Data analysis should include stringent filtering against non-specific binding controls

• Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM):

  • Develop targeted assays for absolute quantification of At5g52605

  • Use isotopically labeled peptide standards matching antibody epitopes

  • Compare antibody-based quantification (ELISA/Western) with MS-based methods

  • Particularly useful for comparing At5g52605 abundance across different plant tissues or conditions

• Post-translational modification (PTM) analysis:

  • Combine antibody enrichment with PTM-specific MS analysis

  • Identify phosphorylation, ubiquitination, or other modifications

  • Correlate modifications with protein activity or localization changes

  • Use complementary antibodies (N-terminal, C-terminal) to ensure complete coverage

• Spatial proteomics:

  • Fractionate plant cells into organelles

  • Use antibodies to track At5g52605 distribution across fractions

  • Combine with mass spectrometry to create comprehensive localization maps

  • Discover potential moonlighting functions in different subcellular compartments

This integrated approach provides multi-dimensional data on At5g52605 function, combining the specificity of antibody-based detection with the comprehensive analysis capabilities of mass spectrometry.

What are the considerations for using At5g52605 antibodies across different plant species?

Cross-species application of At5g52605 antibodies requires careful evaluation of protein conservation and antibody specificity:

• Sequence homology analysis:

  • Align At5g52605 sequences across species of interest

  • Focus on regions recognized by available antibodies (N-terminal, C-terminal, middle region)

  • Predict cross-reactivity based on epitope conservation

• Validation strategies for cross-species applications:

  • Western blot analysis with recombinant proteins from target species

  • Pre-absorption controls with heterologous proteins

  • Side-by-side comparison with species-specific antibodies when available

  • Progressive validation across phylogenetically related species

• Optimization considerations:

  • Adjust extraction protocols for different plant tissues

  • Modify antibody concentration and incubation conditions

  • Consider tissue-specific interfering compounds requiring additional purification steps

When working with non-model species, consider developing custom antibodies targeting the most conserved epitopes of defensin-like proteins or using a combination of antibodies to increase detection probability.