AGD13 Antibody

What is AGD13 and why would researchers need an antibody against it?

AGD13 (Q8LFN9) is a Probable ADP-ribosylation factor GTPase-activating protein found in Arabidopsis thaliana (Mouse-ear cress plant). It functions as a translation product of the AGD13 gene (At4g05330) . Researchers study this protein to understand membrane trafficking and vesicle formation in plant cells, as ADP-ribosylation factor GTPase-activating proteins regulate ARF GTPases involved in these processes. Antibodies against AGD13 are essential tools for detecting, quantifying, and localizing this protein in experimental systems, enabling the study of its expression patterns, protein-protein interactions, and functional roles in plant cell biology .

What are the validated applications for AGD13 antibody?

The commercially available AGD13 antibody (CSB-PA822589XA01DOA) has been validated for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of AGD13 protein in samples

• Western Blot (WB): For detecting the presence and molecular weight of AGD13 protein in complex mixtures

These applications have been validated specifically for ensuring identification of the antigen in Arabidopsis thaliana samples . While other potential applications like immunohistochemistry (IHC) or immunofluorescence (IF) may be possible, researchers should perform their own validation if extending to these methods, as formal validation for these applications was not evident in the available data.

What are the optimal storage conditions for AGD13 antibody?

For maintaining optimal activity of AGD13 antibody:

• Long-term storage: Store at -20°C or -80°C

• Important caution: Avoid repeated freeze-thaw cycles, as these can degrade antibody quality and reduce binding efficacy

• Formulation: The antibody is typically provided in liquid form with 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

For working solutions, maintain at 4°C for short-term use (1-2 weeks). Aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles if multiple experiments are planned over time.

How should AGD13 antibody be validated for specificity in Arabidopsis research?

A comprehensive validation approach for AGD13 antibody specificity should include:

• Positive control testing: Use recombinant Arabidopsis thaliana AGD13 protein as a positive control

• Knockout/knockdown validation: Compare antibody signal between wild-type plants and AGD13 knockout/knockdown lines (such as those created using the amiRNA targeting AGD13, stock number CSHL_056221)

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to block specific binding sites before application in the intended assay

• Cross-reactivity assessment: Test against closely related AGD family proteins (like AGD1, AGD2, AGD3, AGD5, AGD9, AGD15) to ensure specificity

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing the intended target

This multi-faceted approach helps ensure that experimental results reflect the true biology of AGD13 rather than non-specific interactions.

What is the recommended protocol for using AGD13 antibody in Western blotting?

The following optimized protocol is recommended for Western blotting with AGD13 antibody:

Materials needed:

• AGD13 antibody (CSB-PA822589XA01DOA)

• Arabidopsis thaliana protein extract

• Standard Western blotting equipment and reagents

Protocol:

• Sample preparation: Extract proteins from Arabidopsis tissue using a buffer containing protease inhibitors

• Protein separation:

  • Load 20-40 μg protein per lane

  • Separate proteins using SDS-PAGE (10-12% gel recommended)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking:

  • Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20)

  • Incubate for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute AGD13 antibody at 1:500-1:1,000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Washing: Wash membrane 3-4 times (5 minutes each) with TBST

• Secondary antibody incubation:

  • Apply anti-rabbit IgG-HRP at 1:5,000-1:10,000 dilution

  • Incubate for 1 hour at room temperature

• Detection: Use enhanced chemiluminescence (ECL) substrate and visualize using an imaging system

• Expected result: AGD13 appears at approximately 34 kDa

Troubleshooting tip: If background is high, increase washing steps and/or reduce primary antibody concentration to 1:2,000.

How can AGD13 antibody be used for immunoprecipitation of AGD13 and associated proteins?

For effective immunoprecipitation (IP) of AGD13 and its interaction partners:

Materials:

• AGD13 antibody (purified by antigen affinity)

• Protein A/G magnetic or agarose beads

• Plant lysate buffer (containing mild detergents and protease inhibitors)

• Wash and elution buffers

Detailed Protocol:

• Lysate preparation:

  • Homogenize 1-2g Arabidopsis tissue in 3-5ml cold IP buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor cocktail)

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and pre-clear with Protein A/G beads (30 min at 4°C)

• Antibody binding:

  • Incubate 2-5μg of AGD13 antibody with 25-50μl Protein A/G beads for 1 hour at 4°C

  • Wash beads twice with IP buffer

• Immunoprecipitation:

  • Add pre-cleared lysate to antibody-bound beads

  • Incubate overnight at 4°C with gentle rotation

• Washing and elution:

  • Wash beads 4 times with IP buffer

  • Elute proteins with 50μl of 0.1M glycine (pH 2.5) followed by immediate neutralization with 5μl of 1M Tris (pH 8.0), or by boiling in SDS sample buffer

• Analysis:

  • Analyze precipitated proteins by Western blot or mass spectrometry

This protocol is particularly useful for identifying novel interaction partners of AGD13, such as other proteins involved in vesicle trafficking pathways in plants.

How can AGD13 antibody be used to study the subcellular localization of AGD13?

To determine the subcellular localization of AGD13 using immunofluorescence:

Detailed Protocol:

• Sample preparation:

  • Fix Arabidopsis seedlings or protoplasts with 4% paraformaldehyde in PBS for 20 minutes

  • Permeabilize with 0.2% Triton X-100 for 15 minutes

  • Block with 3% BSA in PBS for 1 hour

• Primary antibody incubation:

  • Dilute AGD13 antibody 1:100-1:200 in blocking solution

  • Incubate samples overnight at 4°C

• Washing: Wash samples 3 times with PBS (10 minutes each)

• Secondary antibody incubation:

  • Apply fluorophore-conjugated anti-rabbit secondary antibody (e.g., Alexa Fluor 488) at 1:500 dilution

  • Incubate for 2 hours at room temperature in the dark

• Co-labeling (optional):

  • Include markers for cellular compartments:

    • Golgi: anti-SEC21 or Golgi-tracker dye

    • Endosomes: anti-ARA7/RabF2b

    • Plasma membrane: FM4-64 dye (short incubation time)

• Counterstaining:

  • Stain nuclei with DAPI (1μg/ml) for 10 minutes

• Mounting and imaging:

  • Mount samples in anti-fade mounting medium

  • Image using confocal microscopy

This approach allows researchers to determine where AGD13 localizes within the cell and whether its distribution changes under different experimental conditions, providing insights into its functional role in membrane trafficking.

What approaches can be used to study AGD13 protein-protein interactions with antibody-based techniques?

Several antibody-based techniques can reveal AGD13 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use AGD13 antibody to pull down the protein complex

  • Identify interaction partners via mass spectrometry or Western blotting

  • Critical control: Compare with IgG isotype control IP

• Proximity Ligation Assay (PLA):

  • Use AGD13 antibody with antibodies against suspected interaction partners

  • If proteins are within 40nm, oligonucleotide-linked secondary antibodies enable fluorescent signal amplification

  • Provides spatial information about interactions in situ

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • After identifying candidate interactors, validate with BiFC

  • Compare results with antibody-based methods

• Chromatin Immunoprecipitation (ChIP):

  • If AGD13 functions in transcriptional regulation, use AGD13 antibody for ChIP

  • Sequence precipitated DNA to identify genomic binding sites

• Antibody-based protein arrays:

  • Apply plant lysate to arrays spotted with antibodies against potential interactors

  • Detect bound AGD13 with labeled AGD13 antibody

Each method offers complementary information, with Co-IP providing the most direct evidence of physical interaction under native conditions.

How can AGD13 antibody be used in quantitative studies of AGD13 expression levels?

For quantitative analysis of AGD13 expression levels:

• Quantitative Western Blotting:

  • Use purified recombinant AGD13 protein to create a standard curve

  • Include 3-5 concentration points ranging from 0.1-10ng

  • Process experimental samples alongside standards

  • Use fluorescent secondary antibodies for wider linear range

  • Analysis software: ImageJ with gel analysis tools

• Sandwich ELISA:

  • Coat plate with capture antibody (e.g., commercial AGD13 antibody)

  • Add samples and standards

  • Detect with biotinylated AGD13 antibody or another antibody recognizing a different epitope

  • Standard curve preparation:

    • Prepare 8-point two-fold serial dilution of recombinant AGD13

    • Plot concentration vs. absorbance

    • Use four-parameter logistic regression for curve fitting

  Standard	Concentration (ng/ml)	Mean OD (450nm)
  S1	100	2.458
  S2	50	1.879
  S3	25	1.245
  S4	12.5	0.758
  S5	6.25	0.412
  S6	3.125	0.209
  S7	1.5625	0.105
  S8	0	0.042

• Immunohistochemistry quantification:

  • Use consistent antibody concentrations and development times

  • Analyze signal intensity in defined regions using ImageJ

  • Compare to standard samples with known expression levels

These methods enable researchers to quantitatively measure changes in AGD13 expression across different tissues, developmental stages, or in response to experimental treatments.

What are common issues when using AGD13 antibody and how can they be resolved?

Regularly including positive controls (tissues known to express AGD13) and negative controls (AGD13 knockdown/knockout tissues or pre-immune serum) helps distinguish between technical issues and true biological findings.

How should researchers validate AGD13 antibody for new experimental systems or plant species?

When adapting AGD13 antibody for use in new experimental systems or plant species:

• Sequence homology analysis:

  • Compare AGD13 protein sequence across species

  • Analyze conservation of antibody epitope region

  • Predict cross-reactivity based on sequence identity

  Example analysis for representative plant species:

  Species	% Identity to A. thaliana AGD13	Predicted Cross-Reactivity
  Brassica napus	85-90%	High
  Solanum lycopersicum	65-70%	Moderate
  Oryza sativa	55-60%	Low-Moderate
  Zea mays	50-55%	Low

• Western blot validation:

  • Run protein extracts from multiple species in parallel

  • Include positive control (A. thaliana extract)

  • Confirm expected molecular weight adjustments based on sequence

  • Test different antibody concentrations (1:500, 1:1000, 1:2000)

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP from the new species

  • Confirm target identity by mass spectrometry

  • Assess specificity by analyzing other captured proteins

• Genetic validation:

  • Test antibody in AGD13 knockout/knockdown lines in the new species

  • Use CRISPR/Cas9 or RNAi approaches to generate controls

  • Compare signal between wild-type and modified lines

• Epitope mapping:

  • If cross-reactivity is poor, determine the exact epitope recognized

  • Consider developing new antibodies against conserved regions

This systematic approach ensures scientific rigor when extending AGD13 research to new plant systems.

How can researchers distinguish between different AGD family proteins when using antibodies?

Distinguishing between closely related AGD family proteins requires careful experimental design:

• Comparative sequence analysis:

  • Align protein sequences of all AGD family members

  • Identify regions of high variation as potential specific epitopes

  • Analyze whether the AGD13 antibody epitope overlaps with conserved domains

• Specific detection strategies:

  • Epitope-targeted approach: Use antibodies raised against unique peptide regions

  • Multi-antibody approach: Apply antibodies against different AGD proteins in parallel experiments

  • Molecular weight differentiation: Exploit size differences between AGD proteins

    • AGD13: ~34 kDa

    • Other AGD family members range from ~28-42 kDa

• Knockout/knockdown validation:

  • Use genetic lines with specific AGD genes knocked out/down

  • Test antibody reactivity across these lines

  • True specificity is indicated by signal loss only in AGD13 mutants

• Immunoprecipitation-mass spectrometry:

  • Perform IP with the AGD13 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Identify peptides unique to AGD13 vs. other family members

  • Calculate percent coverage of the target vs. off-targets

• Expression pattern analysis:

  • Compare antibody staining patterns with known expression patterns from transcriptomic data

  • Correlation between antibody signal and mRNA expression supports specificity

This comprehensive approach helps researchers confidently attribute observed signals to AGD13 rather than related proteins.

What statistical approaches are recommended for analyzing quantitative data from AGD13 antibody-based experiments?

For robust statistical analysis of AGD13 antibody-based experimental data:

• Western blot densitometry analysis:

  • Normalization: Always normalize AGD13 band intensity to loading controls (e.g., ACTIN, GAPDH, or total protein stain)

  • Replication: Minimum 3-4 biological replicates

  • Statistical tests:

    • Two conditions: Student's t-test or Mann-Whitney U (non-parametric)

    • Multiple conditions: ANOVA with appropriate post-hoc tests (Tukey's HSD)

  • Visualization: Box plots or bar graphs with individual data points shown

• Immunofluorescence quantification:

  • Sampling strategy: Analyze 10-15 cells across 3+ biological replicates

  • Blinding: Implement analyst blinding to experimental conditions

  • Colocalization analysis: Use Pearson's or Manders' correlation coefficients

  • Statistical comparison: Apply nested ANOVA or mixed models to account for cell-to-cell variability within samples

• ELISA data analysis:

  • Standard curve fitting: Use four-parameter logistic regression (4PL)

  • Sample interpolation: Report with 95% confidence intervals

  • Quality control metrics:

    • Include coefficient of variation (CV) < 15%

    • Monitor lower limit of quantification (LLOQ)

  • Assay validation: Document linearity, recovery, and parallelism

• Addressing potential pitfalls:

  • Antibody saturation: Verify working in linear range of detection

  • Signal normalization: Use standard curves for absolute quantification

  • Batch effects: Include control samples across experimental batches

• Data sharing best practices:

  • Report all raw data and detailed methodological parameters

  • Include representative images of entire blots/gels

  • Share analysis code and image processing workflows

Following these guidelines ensures reliable quantitative interpretation of AGD13 protein levels and localization.

How might AGD13 antibodies be combined with emerging technologies to advance plant molecular biology research?

Several innovative approaches combine AGD13 antibodies with cutting-edge technologies:

• Proximity proteomics with AGD13 antibodies:

  • Use AGD13 antibodies conjugated to engineered ascorbate peroxidase (APEX)

  • Apply to living plant cells to biotinylate proteins in proximity to AGD13

  • Analyze the AGD13 proximal proteome via streptavidin pulldown and mass spectrometry

  • This approach maps the dynamic protein neighborhood of AGD13 in vivo

• Super-resolution microscopy applications:

  • Apply AGD13 antibodies with STORM/PALM super-resolution techniques

  • Achieve 20-30nm resolution of AGD13 localization in plant endomembrane systems

  • Track AGD13 clustering and distribution relative to membrane microdomains

  • Compare results with conventional confocal microscopy to reveal previously undetected organizational principles

• Microfluidic antibody-based single-cell analysis:

  • Integrate AGD13 antibody detection into plant single-cell analysis platforms

  • Quantify AGD13 protein levels in individual protoplasts alongside transcriptome analysis

  • Map cell-to-cell heterogeneity in AGD13 expression across tissues

• CRISPR-based tagging combined with antibody validation:

  • Use CRISPR/Cas9 to add epitope tags to endogenous AGD13

  • Compare commercial antibody performance with anti-tag antibodies

  • Develop improved validation strategies for plant cell biology

• Multispectral tissue imaging:

  • Apply AGD13 antibodies in multiplexed immunofluorescence panels

  • Simultaneously visualize AGD13 alongside 5-7 other proteins in the same tissue section

  • Map spatial relationships between AGD13 and other components of membrane trafficking machinery

These approaches represent the frontier of plant molecular biology research using AGD13 antibodies, enabling unprecedented insights into protein function and cellular organization.

What are the challenges in developing AGD13 antibodies against specific post-translational modifications?

Developing antibodies against post-translationally modified (PTM) AGD13 presents several challenges:

• Identification of relevant modifications:

  • Current knowledge gap: PTMs of AGD13 remain largely uncharacterized

  • Approach: Perform phosphoproteomic, glycoproteomic, and ubiquitinomic analyses of purified AGD13

  • Challenge: Low abundance of modified forms requires enrichment strategies

• Antibody development considerations:

  • Immunogen design:

    • Synthesize peptides containing the specific modification

    • Include carrier proteins that preserve the modification during immunization

    • Design peptides with the modification centrally positioned

  • PTM stability issues:

    • Phosphorylation: Relatively stable but can be lost during processing

    • Ubiquitination: Complex topology with multiple potential linkages

    • Glycosylation: Complex structures with potential microheterogeneity

  • Validation requirements:

    • Test against modified and unmodified recombinant proteins

    • Verify with modification-eliminating treatments (phosphatases, deglycosylating enzymes)

• Specificity challenges:

  • Cross-reactivity: Ensure antibodies recognize the modification in the context of AGD13 sequence

  • Background issues: PTM-specific antibodies often show higher background than total protein antibodies

  • Controls needed: Include tissues/cells treated with PTM inhibitors as negative controls

• Quantification complexities:

  • Stoichiometry determination: Develop strategies to determine what fraction of total AGD13 carries the modification

  • Dynamic range: Modified forms may represent <1% of total protein

  • Method development: Consider developing targeted mass spectrometry assays as complementary approaches

• Biological relevance assessment:

  • Functional impact: Determine how modification affects AGD13's GTPase-activating activity

  • Regulatory significance: Map conditions that alter modification status

  • Evolutionary conservation: Compare PTM sites across plant species

Addressing these challenges will enable researchers to understand the regulatory mechanisms controlling AGD13 function in plants.

How does the development of plant antibodies like anti-AGD13 compare with antibody development for mammalian systems?

The development of plant-specific antibodies presents unique challenges and considerations compared to mammalian systems:

For AGD13 antibody development specifically, researchers should:

• Consider using plant-feeding arthropods (rather than mammals) for immunization to potentially improve immunogenicity

• Develop comprehensive validation panels including multiple plant species

• Optimize extraction protocols specifically for membrane-associated plant proteins

• Establish plant-specific positive and negative controls for each application

These considerations highlight why plant molecular biology often requires specialized expertise in antibody development and application.

What can researchers learn from studies using ADAMTS13 antibodies that might apply to AGD13 antibody research?

Although ADAMTS13 (a metalloprotease involved in blood clotting) and AGD13 (a plant ADP-ribosylation factor GTPase-activating protein) have different functions and occur in different organisms, several methodological insights from ADAMTS13 antibody research can inform AGD13 studies:

• Antibody characterization approaches:

  • ADAMTS13 research demonstrates rigorous antibody validation through multiple methodologies

  • Application to AGD13: Implement similar multi-method validation including epitope mapping, specificity testing against related proteins, and correlation with genetic knockdown

• Epitope mapping strategies:

  • ADAMTS13 studies identified specific binding epitopes through systematic approaches

  • Application to AGD13: Employ linear epitope mapping using overlapping peptides to precisely identify antibody binding regions, which helps predict cross-reactivity with related AGD proteins

• Subclass distribution analysis:

  • ADAMTS13 research characterized IgG subclasses of antibodies which correlated with different functions

  • Application to AGD13: When developing monoclonal antibodies, analyze IgG subclass distribution to select optimal antibodies for specific applications (IgG1 may be preferable for some applications, while IgG4 for others)

• Functional correlation with antibody binding:

  • ADAMTS13 studies correlated antibody binding with functional inhibition

  • Application to AGD13: Develop assays that directly measure AGD13's GTPase-activating function to determine if antibody binding affects protein activity

• Quantitative assay development:

  • ADAMTS13 research established reliable quantitative assays for both protein and antibodies

  • Application to AGD13: Develop standardized quantitative assays with defined reference ranges for AGD13 protein levels in different plant tissues and developmental stages

• Detection in complex matrices:

  • ADAMTS13 research successfully detected the protein in complex biological fluids

  • Application to AGD13: Optimize extraction conditions to reliably detect AGD13 in complex plant tissue extracts with potential interfering compounds