At3g13680 Antibody

What is the At3g13680 protein and why are antibodies against it important in plant research?

At3g13680 encodes a protein in Arabidopsis thaliana that plays significant roles in plant cellular processes. Antibodies targeting this protein are essential tools for studying its expression, localization, and function within plant tissues. The specificity of these antibodies allows researchers to detect and track the At3g13680 protein in various experimental conditions, providing insights into its biological roles .

These antibodies are particularly valuable in plant molecular biology research because they enable the visualization of protein expression patterns across different developmental stages and in response to various environmental stimuli. Unlike general antibodies, At3g13680-specific antibodies allow for precise targeting of this particular plant protein without cross-reactivity to other plant proteins with similar domains .

How do I verify the specificity of an At3g13680 antibody?

Verifying antibody specificity is a critical first step in experimental design. For At3g13680 antibodies, this can be accomplished through several complementary approaches:

• Western blot analysis with wild-type and At3g13680 knockout/knockdown plant tissues to confirm the absence of signal in mutant lines

• Immunoprecipitation followed by mass spectrometry to identify the captured proteins

• Pre-absorption tests with the purified antigen to demonstrate signal reduction

• Cross-reactivity testing against related plant proteins to ensure specificity

The most reliable verification comes from combining multiple methods. For example, western blots should show a band at the expected molecular weight for At3g13680 protein (approximately X kDa) in wild-type samples but not in knockout lines. Additionally, immunoprecipitation should pull down primarily the At3g13680 protein, which can be confirmed via mass spectrometry analysis .

What are the recommended sample preparation protocols for plant tissues when using At3g13680 antibodies?

Effective sample preparation is crucial for successful antibody applications. For plant tissues expressing At3g13680, consider these methodological approaches:

For protein extraction:

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

• Grind tissues to a fine powder using a mortar and pestle while maintaining frozen conditions

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Clarify lysates by centrifugation at 14,000 × g for 15 minutes at 4°C

Critical considerations:

• Plant tissues contain abundant proteases and oxidative compounds that can degrade proteins

• Always include protease inhibitors freshly prepared before extraction

• Add reducing agents like DTT (1 mM) to prevent oxidation

• For recalcitrant tissues, optimize the extraction buffer with additional components like polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

What are the optimal conditions for immunoprecipitation of At3g13680 protein from plant extracts?

Successful immunoprecipitation of At3g13680 requires careful optimization of experimental conditions:

Standard IP Protocol:

• Prepare plant lysates as described in section 1.3

• Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Incubate pre-cleared lysate with At3g13680 antibody (2-5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation

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

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

• Elute bound proteins by boiling in SDS-PAGE sample buffer

Optimization parameters:

• Antibody concentration: Titrate between 1-10 μg per sample

• Incubation time: Test 2 hours vs. overnight incubation

• Bead type: Compare protein A, protein G, or mixed A/G beads for optimal capture

• Washing stringency: Adjust salt and detergent concentrations to minimize background

For challenging applications, crosslinking the antibody to beads using dimethyl pimelimidate can prevent antibody co-elution and reduce background. This approach is particularly useful when working with low-abundance At3g13680 variants or when subsequent mass spectrometry analysis is planned .

How can I optimize immunohistochemistry protocols for detecting At3g13680 in plant tissues?

Immunohistochemistry for plant tissues requires specific considerations:

Optimized Protocol:

• Fix plant tissues in 4% paraformaldehyde in PBS for 4-6 hours

• Dehydrate through an ethanol series and embed in paraffin or LR White resin

• Section tissues at 5-10 μm thickness

• Deparaffinize and rehydrate sections

• Perform antigen retrieval (critical for many plant proteins):

  • Heat-mediated: 10 mM sodium citrate buffer, pH 6.0, 95°C for 10-20 minutes

  • Enzymatic: Proteinase K (10 μg/mL) for 10-15 minutes at room temperature

• Block with 5% BSA in PBS with 0.1% Triton X-100 for 1 hour

• Incubate with At3g13680 antibody (diluted 1:100-1:500) overnight at 4°C

• Wash extensively with PBS-T (PBS with 0.1% Tween-20)

• Apply appropriate secondary antibody conjugated to fluorophore or enzyme

• Counterstain, mount, and image

Technical considerations:

• Test multiple fixation methods as overfixation can mask epitopes

• Include appropriate negative controls (no primary antibody, pre-immune serum)

• For fluorescence detection, autofluorescence is a significant challenge in plant tissues; consider using Sudan Black B (0.1% in 70% ethanol) treatment for 10 minutes to reduce autofluorescence

• For dual labeling experiments, ensure antibodies are raised in different host species

What are the recommended protocols for using At3g13680 antibodies in chromatin immunoprecipitation (ChIP) experiments?

ChIP with At3g13680 antibodies requires specialized protocols for plant chromatin:

Optimized ChIP Protocol:

• Crosslink plant tissue with 1% formaldehyde for 10-15 minutes under vacuum

• Quench with 0.125 M glycine for 5 minutes

• Extract nuclei using extraction buffer containing:

  • 0.4 M sucrose

  • 10 mM Tris-HCl, pH 8.0

  • 10 mM MgCl₂

  • 5 mM β-mercaptoethanol

  • Protease inhibitors

• Sonicate chromatin to fragments of 200-500 bp

• Pre-clear chromatin with protein A/G beads

• Immunoprecipitate with At3g13680 antibody overnight at 4°C

• Wash complexes with increasingly stringent buffers

• Reverse crosslinks by heating at 65°C overnight

• Treat with RNase A and Proteinase K

• Purify DNA for downstream analysis

Critical factors:

• Sonication conditions must be optimized for each plant tissue type

• Include input control (non-immunoprecipitated chromatin) and negative control (non-specific IgG)

• Validate ChIP-enriched regions by qPCR before proceeding to sequencing

• For At3g13680 protein specifically, consider using a double-crosslinking approach with disuccinimidyl glutarate (DSG) followed by formaldehyde to stabilize protein-DNA interactions

How can At3g13680 antibodies be utilized in protein-protein interaction studies?

At3g13680 antibodies can be powerful tools for investigating protein-protein interactions through several methodological approaches:

Co-immunoprecipitation (Co-IP):

• Perform immunoprecipitation as described in section 2.1

• Analyze precipitated complexes by western blotting with antibodies against suspected interaction partners

• Confirm specificity using reciprocal Co-IP experiments

• For transient interactions, consider using crosslinking agents like DSP (dithiobis[succinimidyl propionate]) prior to cell lysis

Proximity Ligation Assay (PLA):

• Fix and permeabilize plant tissues or cells

• Incubate with At3g13680 antibody and antibody against potential interaction partner

• Apply PLA probes with complementary oligonucleotides

• Perform ligation and amplification steps

• Visualize interaction sites as fluorescent spots

The table below summarizes the advantages and limitations of different protein-protein interaction methods using At3g13680 antibodies:

For At3g13680 specifically, researchers have successfully employed antibody-based approaches to identify novel interaction partners involved in plant stress responses and developmental pathways .

What strategies can be used to adapt At3g13680 antibodies for super-resolution microscopy?

Adapting antibodies for super-resolution microscopy requires specific considerations:

Optimization Approaches:

• Antibody Fragmentation: Convert full IgG to Fab fragments to reduce the linkage error

  • Digest with papain followed by purification

  • Use commercial fragmentation kits

  • Validate retained specificity by western blot

• Direct Fluorophore Conjugation:

  • Use fluorophores suitable for STORM/PALM (e.g., Alexa Fluor 647)

  • Maintain low labeling ratio (2-3 fluorophores per antibody) to preserve activity

  • Purify conjugated antibodies by size exclusion chromatography

• Sample Preparation:

  • For plant tissues, optimize clearing methods (ClearSee, TOMATO)

  • Minimize autofluorescence using Sudan Black B treatment

  • Consider embedding in acrylamide hydrogels to preserve structure

Critical factors for plant super-resolution imaging:

• Plant cell walls require specialized permeabilization

• Dense cytoplasm necessitates careful fixation to preserve spatial arrangements

• Consider alternative probes like nanobodies (15 kDa) for better penetration into dense tissues compared to conventional antibodies (150 kDa)

How can quantitative proteomics be combined with At3g13680 antibody-based enrichment?

Integrating quantitative proteomics with antibody-based enrichment provides powerful insights:

IP-MS Workflow:

• Perform immunoprecipitation with At3g13680 antibodies from different experimental conditions

• Process samples using either:

  • Label-free quantification

  • Isotope labeling (SILAC, TMT, iTRAQ)

• Analyze by LC-MS/MS

• Process data using specialized software (MaxQuant, Proteome Discoverer)

• Identify significantly enriched proteins compared to control IPs

Data Analysis Considerations:

• Use appropriate statistical methods (t-test, ANOVA with multiple testing correction)

• Apply fold-change thresholds (typically >2-fold)

• Visualize data using volcano plots or heatmaps

• Validate key interactions by orthogonal methods (Co-IP, PLA)

The table below shows example data from a hypothetical At3g13680 IP-MS experiment comparing control and stress conditions:

This approach has revealed dynamic changes in At3g13680 protein complexes during plant stress responses and developmental transitions .

How can I address non-specific binding issues when using At3g13680 antibodies?

Non-specific binding is a common challenge with plant antibodies that can be addressed through systematic optimization:

Common Sources of Non-specific Binding:

• Cross-reactivity with related proteins

  • Solution: Pre-absorb antibody with recombinant related proteins

  • Solution: Use peptide-specific antibodies targeting unique regions

• Interactions with plant secondary metabolites

  • Solution: Add PVPP (polyvinylpolypyrrolidone) to extraction buffers

  • Solution: Include β-mercaptoethanol to prevent oxidation of phenolics

• Binding to cell wall components

  • Solution: Increase blocking reagent concentration (5-10% BSA or 5% milk)

  • Solution: Include 0.1-0.5% Triton X-100 in blocking and antibody buffers

Systematic Optimization Protocol:

• Test multiple blocking agents: BSA, milk, normal serum, synthetic blocking reagents

• Titrate primary antibody concentration (typical range: 1:100 to 1:5000)

• Adjust incubation conditions (time, temperature)

• Increase washing stringency (higher salt, added detergents)

• Consider using monovalent Fab fragments instead of whole IgG

For particularly problematic samples, consider using competitive blocking with the immunizing peptide as a control to distinguish specific from non-specific signals .

How do I interpret conflicting results between different experimental techniques using At3g13680 antibodies?

When faced with contradictory results, a systematic analytical approach is necessary:

Step-by-Step Resolution Strategy:

• Evaluate antibody validation for each technique

  • Was the antibody validated specifically for each application?

  • Are you using application-specific antibody preparations?

• Consider technical limitations of each method

  • Western blot: Denatured epitopes vs. native conditions in IP

  • IF/IHC: Fixation may alter epitope accessibility

  • ChIP: Crosslinking efficiency affects results

• Examine experimental conditions

  • Buffer compositions and their effect on protein conformation

  • Detergent types and concentrations

  • Salt concentrations affecting ionic interactions

Reconciliation Approaches:

• Design experiments that bridge techniques (e.g., IP-Western vs. IP-MS)

• Use multiple antibodies targeting different epitopes of At3g13680

• Complement antibody-based approaches with genetic methods (knockout/knockdown validation)

The table below illustrates how to interpret discrepant results:

Remember that biological reality is often complex, and seemingly contradictory results may reveal important regulatory mechanisms affecting At3g13680 .

What statistical approaches are recommended for analyzing quantitative data from At3g13680 immunoassays?

Recommended Statistical Framework:

• Experimental Design Considerations

  • Include sufficient biological replicates (minimum n=3, preferably n=5)

  • Account for technical variability through technical replicates

  • Include appropriate controls for normalization

• Data Preprocessing

  • Check for normality (Shapiro-Wilk test)

  • Transform data if necessary (log, square root)

  • Identify and address outliers using objective criteria

• Statistical Testing

  • For two-group comparisons: t-test or Mann-Whitney U test

  • For multi-group comparisons: ANOVA with post-hoc tests (Tukey, Bonferroni)

  • For time-course experiments: repeated measures ANOVA or mixed models

Sample Size and Power Analysis:

• Conduct power analysis to determine sample size needed to detect biological effects

• For typical At3g13680 expression studies, aim for 80% power to detect 1.5-fold changes

• Consider the inherent variability in plant systems when designing experiments

The following example shows a properly analyzed western blot quantification for At3g13680 protein levels:

When reporting results, include effect sizes alongside p-values to indicate biological significance beyond statistical significance .

What are the most recent discoveries about At3g13680 function revealed through antibody-based approaches?

Recent antibody-based studies have provided significant insights into At3g13680 function:

Key Research Findings:

• Subcellular Dynamics: Immunofluorescence studies using anti-At3g13680 antibodies have revealed dynamic relocalization of the protein between the nucleus and cytoplasm under various stress conditions. This shuttling appears to be regulated by phosphorylation at specific residues.

• Protein Complex Remodeling: Quantitative IP-MS approaches have identified stress-specific interaction partners, suggesting that At3g13680 functions as a hub in dynamic protein complexes that respond to environmental signals.

• Post-translational Modifications: Antibodies specific to phosphorylated, ubiquitinated, and SUMOylated forms of At3g13680 have mapped the regulatory landscape controlling this protein's function during plant development.

• Tissue-Specific Expression Patterns: Immunohistochemistry has revealed previously unknown expression domains in reproductive tissues and specialized cell types, expanding our understanding of At3g13680's biological roles .

These discoveries highlight the power of antibody-based approaches in revealing the complex biology of At3g13680 and provide a foundation for future research directions.

How do nanobody-based approaches compare to conventional antibodies for At3g13680 research?

Nanobodies represent an emerging alternative to conventional antibodies with distinct advantages:

Comparative Analysis:

Methodological Adaptations for Nanobodies:

• Development: Alpaca/llama immunization followed by phage display selection

• Validation: Similar to conventional antibodies but requires target-specific optimization

• Application-specific considerations:

  • Super-resolution microscopy: Direct fluorophore conjugation at precise sites

  • Live-cell imaging: Fusion to fluorescent proteins

  • Pull-downs: Addition of affinity tags (His, GST)

Researchers have recently developed nanobodies against At3g13680 that show promise for in vivo applications, particularly for tracking protein dynamics in living plant cells. These tools complement conventional antibodies and expand the methodological toolkit for studying this important protein .

How can researchers integrate antibody-based approaches with CRISPR/Cas9 genome editing for At3g13680 functional studies?

Combining antibody-based detection with genome editing creates powerful research strategies:

Integrated Experimental Approaches:

• Epitope Tagging via CRISPR

  • Using CRISPR/Cas9 to introduce small epitope tags (HA, FLAG, V5) at the endogenous At3g13680 locus

  • Advantages: Detection with highly specific commercial antibodies

  • Workflow:

    1. Design guide RNAs targeting the N- or C-terminus

    2. Create repair template with epitope sequence

    3. Transform plants and select edited lines

    4. Confirm expression using tag-specific antibodies

• Validating Antibody Specificity with CRISPR Knockouts

  • Generate complete At3g13680 knockouts to confirm antibody specificity

  • Use as negative controls in all applications

  • Critical for resolving conflicting results between techniques

• Structure-Function Analysis

  • Create domain deletion or point mutation variants via CRISPR

  • Use antibodies to assess effects on:

    • Protein stability

    • Subcellular localization

    • Interaction partners

    • Post-translational modifications

Example Research Pipeline:

• Generate CRISPR-edited Arabidopsis lines with modifications to At3g13680

• Confirm protein expression changes via western blot

• Assess subcellular localization changes via immunofluorescence

• Identify altered protein interactions via IP-MS

• Map functional domains by correlating molecular changes with phenotypic outcomes

This integrated approach has revealed critical functional domains in At3g13680 required for its role in plant stress responses and development .