At3g04903 Antibody

What is At3g04903 and why are antibodies against it important in plant research?

At3g04903 is a gene located on chromosome 3 of Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology . Antibodies targeting the protein encoded by this gene enable researchers to study its expression, localization, and function within plant tissues. These antibodies are particularly valuable for investigating protein-protein interactions, protein modifications, and cellular localization patterns that cannot be determined through genomic or transcriptomic approaches alone .

What types of antibodies are available for At3g04903 protein detection?

Based on available research tools, antibodies against At3g04903 protein are typically polyclonal or monoclonal antibodies developed specifically for research applications . These antibodies are generally available in two formats:

• Standard antibody preparations (typically 2ml)

• Concentrated antibody preparations (typically 0.1ml)

Polyclonal antibodies offer broader epitope recognition but may have batch-to-batch variability, while monoclonal antibodies provide consistent specificity but may be limited to single epitope recognition.

How should At3g04903 antibodies be stored and handled in laboratory settings?

For optimal performance and longevity, At3g04903 antibodies should follow storage protocols similar to other plant protein antibodies:

• Short-term storage (less than 1 month): 4°C

• Long-term storage (more than 1 month): -80°C

• Avoid repeated freeze-thaw cycles

• When shipping is required, cold packs should be used

These storage conditions help maintain antibody functionality by preventing protein denaturation and preserving epitope recognition capabilities.

What are the recommended validation methods for At3g04903 antibodies before experimental use?

Before using At3g04903 antibodies in critical experiments, several validation steps should be performed:

• Western blot analysis using:

  • Microsomal fractions from wild-type A. thaliana leaves

  • Membrane fractions from transgenic systems expressing the protein

  • Appropriate negative controls (knockout or mutant lines)

• Immunoprecipitation followed by mass spectrometry (IP-MS) to confirm:

  • Specific binding to the target protein

  • Identification of potential protein interaction partners

• Immunolocalization studies comparing:

  • Wild-type plants

  • Knockout/mutant plants

  • Plants with altered expression of the target protein

These validation approaches ensure that observed signals are specific to the target protein rather than resulting from non-specific binding.

What experimental techniques are most effective for using At3g04903 antibodies?

At3g04903 antibodies can be employed in multiple experimental techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay)

  • Allows quantitative measurement of protein levels

  • Suitable for high-throughput screening

  • Primary application listed for similar plant antibodies

• Western Blotting

  • Provides information about protein size and possible modifications

  • Requires optimization of blocking conditions for plant proteins

  • Can utilize horseradish peroxidase (HRP) conjugated secondary antibodies

• Immunoprecipitation

  • Valuable for studying protein-protein interactions

  • Can be coupled with mass spectrometry (IP-MS)

  • May reveal novel biological functions and pathways

• Immunohistochemistry/Immunocytochemistry

  • Reveals subcellular localization of the protein

  • Can track protein redistribution upon stimuli

  • May require specific fixation protocols for plant tissues

How can genetic immunization approaches be applied to generate improved At3g04903 antibodies?

Genetic immunization represents an advanced approach for generating antibodies against plant membrane proteins like those encoded by At3g04903:

• This technique involves immunizing animals with DNA encoding the target protein rather than with purified protein or peptides.

• The approach is particularly valuable for membrane proteins that are difficult to purify in their native conformation.

• The method has been successfully demonstrated for generating antibodies against KAT1, another Arabidopsis membrane protein of low abundance .

Implementation steps include:

• Cloning the At3g04903 coding sequence into an appropriate expression vector

• Administering the DNA construct to animals through intramuscular injection

• Screening resultant antibodies against membrane fractions from wild-type and mutant plants

This approach avoids "the time and labour consuming purification of native or recombinant proteins and peptides usually necessary for conventional immunisation techniques" .

How can At3g04903 antibodies be employed in studying protein-protein interactions and complex formation?

Investigating protein-protein interactions using At3g04903 antibodies involves several sophisticated approaches:

• Co-immunoprecipitation (Co-IP) experiments:

  • Express tagged versions of At3g04903 protein (e.g., FLAG-tagged, HA-tagged)

  • Perform pull-down experiments using antibodies against the tag

  • Identify co-precipitating proteins through western blotting or mass spectrometry

  • Confirm interactions through reciprocal Co-IP experiments

• In vitro interaction studies:

  • Express and purify GST-tagged potential interaction partners

  • Incubate with At3g04903 protein purified from Arabidopsis

  • Perform GST pull-down assays to confirm direct interactions

• In vivo confirmation in F1 plants:

  • Generate plants expressing both At3g04903 and potential interactors with different tags

  • Perform Co-IP experiments to validate interactions in physiological context

What approaches can address epitope masking or conformational changes when working with At3g04903 antibodies?

Research with plant antibodies indicates that protein conformation and epitope accessibility can significantly impact antibody recognition:

• Cryptic epitope exposure:

  • Some plant protein epitopes are only exposed after specific post-translational modifications

  • For example, certain epitopes may only become accessible after cleavage and pyroglutamylation

  • Consider testing samples under conditions that might expose cryptic epitopes (e.g., partial denaturation, enzymatic treatment)

• Conformational epitope recognition:

  • Some antibodies recognize three-dimensional structures rather than linear sequences

  • Denaturation conditions in western blotting may destroy these epitopes

  • Native-PAGE or immunoprecipitation may be required for detection

• Engineered antibodies with pH-dependent binding:

  • Advanced "sweeping antibodies" with pH-dependent antigen binding can improve antigen detection and clearance

  • These approaches might be adapted for plant research applications

How can At3g04903 antibodies be integrated into multi-omics approaches for comprehensive plant biology studies?

Integration of antibody-based approaches with other omics techniques provides more comprehensive insights:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • If At3g04903 encodes a DNA-binding protein or chromatin-associated factor

  • Identify genomic binding sites and target genes

  • Construct libraries using appropriate systems (e.g., Ovation Ultralow DR Multiplex System)

• Correlation with transcriptomic data:

  • Combine protein detection using At3g04903 antibodies with RNA-seq

  • Analyze whether protein levels correlate with transcript abundance

  • Identify post-transcriptional regulatory mechanisms

• Glycomics integration:

  • If At3g04903 protein undergoes glycosylation, combine antibody detection with glycan profiling

  • Use specialized antibodies that recognize specific glycan structures (like those in arabinogalactan proteins)

• Metabolomics correlation:

  • Investigate relationships between At3g04903 protein levels and metabolite profiles

  • Particularly relevant if the protein functions in metabolic pathways or signaling

What are common challenges when using At3g04903 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with plant protein antibodies:

• High background in western blots:

  • Solution: Optimize blocking conditions (try different blockers like 5% BSA or 5% non-fat milk)

  • Increase washing stringency (higher salt concentration or mild detergents)

  • Dilute primary antibody further

  • Use more specific secondary antibodies

• Weak or no signal detection:

  • Solution: Enrich for the protein fraction where At3g04903 is expected (membrane fraction, nuclear extract, etc.)

  • Confirm protein expression timing and conditions

  • Try different extraction buffers that better preserve protein structure

  • Consider alternative detection methods (chemiluminescence vs. fluorescence)

• Multiple bands or unexpected band sizes:

  • Solution: Verify with knockout/mutant controls

  • Consider post-translational modifications or alternative splicing

  • Test specificity with peptide competition assays

  • Optimize SDS-PAGE conditions

How can experimental controls be designed to validate At3g04903 antibody specificity in plant research?

Proper controls are essential for confirming antibody specificity:

• Genetic controls:

  • Compare signal between wild-type plants and At3g04903 knockout/mutant plants

  • Use overexpression lines to confirm increased signal intensity

  • Test in heterologous expression systems (e.g., yeast, tobacco)

• Biochemical controls:

  • Pre-absorb antibody with purified antigen or immunizing peptide

  • Include isotype control antibodies with the same species origin but irrelevant specificity

  • Use secondary antibody-only controls to identify non-specific binding

• Technical controls:

  • Include loading controls (e.g., anti-tubulin, anti-H3 for nuclear proteins)

  • Use molecular weight markers to confirm expected protein size

  • Process samples in parallel with well-characterized antibodies

What considerations are important when analyzing post-translational modifications of At3g04903 protein?

Detecting post-translational modifications (PTMs) requires specialized approaches:

• Histone modification analysis:

  • If At3g04903 interacts with histones or chromatin, specific antibodies against histone modifications may be needed

  • Examples include H3K9ac, H3K27ac, H3ac for acetylation studies

  • Multiple antibodies may be required to characterize different modifications

• Phosphorylation detection:

  • Use phospho-specific antibodies if available

  • Alternatively, treat samples with phosphatases and observe mobility shifts

  • Consider Phos-tag™ gels for enhanced resolution of phosphorylated proteins

• Glycosylation analysis:

  • Plant proteins often undergo unique glycosylation patterns

  • Specialized antibodies against specific glycan structures may be required

  • Enzymatic deglycosylation can help identify glycosylated forms

How can At3g04903 antibodies contribute to understanding developmental and immune-related processes in plants?

Antibodies against plant proteins have revealed important insights into developmental regulation and immune responses:

• Flowering time regulation:

  • If At3g04903 is involved in flowering pathways, antibodies can track protein expression changes during the transition from vegetative to reproductive growth

  • Compare protein levels between long-day and short-day conditions

  • Analyze protein modification patterns during developmental transitions

• Pathogen response studies:

  • Monitor protein relocalization during pathogen challenge

  • Track accumulation patterns at specific cellular locations (e.g., peripheral lobes of leaf epidermal cells)

  • Investigate protein-protein interactions that form during immune responses

• Signal transduction analysis:

  • Examine how At3g04903 protein levels or modifications respond to hormonal treatments

  • Study protein complex formation during signaling events

  • Identify post-translational modifications that occur during signaling

What emerging antibody technologies might enhance research with At3g04903 protein?

Several cutting-edge antibody technologies could benefit plant protein research:

• Engineered antibodies with enhanced properties:

  • "Sweeping antibodies" with pH-dependent binding for improved detection sensitivity

  • Antibodies designed for intracellular expression ("intrabodies") to track proteins in living cells

  • Nanobodies derived from camelid antibodies for improved penetration of plant tissues

• Alternative antibody isotypes:

  • While most research antibodies are IgG, other isotypes like IgM may offer advantages

  • IgM antibodies can provide higher avidity through multiple binding sites

  • This can be particularly valuable for targets with multiple epitopes or repeating structures

• Bispecific antibodies:

  • Antibodies engineered to recognize two different epitopes

  • Could simultaneously target At3g04903 and an interaction partner

  • Would allow tracking of protein complexes rather than individual proteins

How can computational approaches improve At3g04903 antibody design and application?

Computational tools can enhance antibody-based research in several ways:

• Epitope prediction and antibody design:

  • In silico analysis of At3g04903 protein structure to identify optimal epitopes

  • Prediction of surface-exposed regions more likely to be accessible to antibodies

  • Design of antibodies with improved specificity based on structural information

• Integration with protein interaction networks:

  • Prediction of potential interaction partners to guide Co-IP experiments

  • Analysis of protein domains to understand functional relationships

  • Network-based approaches to position At3g04903 in biological pathways

• Multiscale modeling:

  • Simulation of antibody-antigen interactions at molecular level

  • Understanding how antibody isotype affects binding properties

  • Predicting effects of post-translational modifications on antibody recognition

Table of Experimental Applications for At3g04903 Antibody

Comparative Analysis of Antibody Generation Methods for Plant Proteins