At3g51990 Antibody

What is At3g51990 and why is it important in plant research?

At3g51990 refers to a serine/threonine-protein kinase-like protein in Arabidopsis thaliana. This protein belongs to a significant family of regulatory enzymes that play crucial roles in cellular signaling pathways in plants.

The importance of this protein stems from its involvement in:

• Signal transduction pathways related to stress responses

• Potential roles in plant development and growth regulation

• Evolutionary conservation across plant species

According to available data, homologs of this protein exist in various plant species, including Amborella trichopoda (LOC18449076), which is considered a basal angiosperm that can provide insights into early flowering plant evolution .

What types of antibodies are available for detecting At3g51990?

Researchers have two main approaches for developing antibodies against At3g51990:

• Peptide antibodies: Generated against short synthetic peptides (typically 10-15 amino acids) designed from unique regions of At3g51990. These have shown limited success rates in plant protein studies.

• Recombinant protein antibodies: Produced using larger protein fragments expressed in bacterial systems.

Research data comparing antibody generation methods shows significant differences in success rates:

For the most reliable results, recombinant protein antibodies with affinity purification are recommended based on comprehensive studies of Arabidopsis protein detection .

How should researchers validate At3g51990 antibodies before experimental use?

Proper validation of At3g51990 antibodies is essential for reliable experimental results. A systematic validation approach should include:

• Western blot analysis:

  • Test against wild-type Arabidopsis protein extracts

  • Include a negative control using at3g51990 knockout/knockdown mutants

  • Verify correct molecular weight (~110 kDa expected)

• Specificity testing:

  • Cross-reactivity assessment against related kinases

  • Testing in multiple tissue types to confirm consistent detection

• Functional validation:

  • Immunoprecipitation followed by mass spectrometry

  • Blocking experiments with immunizing peptide/protein

As noted in Arabidopsis antibody resource studies, proper affinity purification significantly improves antibody performance. For example, crude antisera often fail to provide signals in immunolocalization, while affinity-purified antibodies show dramatically improved detection rates .

What are the optimal experimental design considerations when using At3g51990 antibodies for subcellular localization studies?

When designing subcellular localization experiments using At3g51990 antibodies, researchers should implement the following methodological approaches:

• Sample preparation optimization:

  • Fresh tissue fixation with 4% paraformaldehyde

  • Careful cell wall permeabilization (enzymatic or mechanical)

  • Antigen retrieval optimization for embedded sections

• Controls implementation:

  • Positive control: Co-localization with known compartment markers

  • Negative control: Parallel experiments with pre-immune serum

  • Genetic control: Analysis of null mutant tissues

• Signal validation approach:

  • Use of multiple antibodies raised against different epitopes

  • Correlation with GFP-fusion protein localization data

  • Verification with biochemical fractionation approaches

From published data on Arabidopsis protein localization, successful immunocytochemistry requires testing multiple fixation protocols and antibody concentrations (typically starting at 1:500 dilution for immunolocalization) . Include subcellular marker antibodies (e.g., BiP for ER, γ-COP for Golgi) to provide context for At3g51990 localization .

How can researchers overcome technical challenges in generating specific antibodies against plant serine/threonine kinases like At3g51990?

Generating specific antibodies against plant kinases presents several challenges due to sequence conservation within kinase families. Advanced methodological solutions include:

• Epitope selection strategies:

  • Bioinformatic analysis to identify unique regions outside the conserved kinase domain

  • Consideration of protein tertiary structure to select exposed regions

  • Sliding window approach to obtain regions with <40% sequence similarity to related kinases

• Expression system optimization:

  • Use of E. coli strains optimized for disulfide bond formation

  • Controlled induction conditions to minimize inclusion body formation

  • Expression of protein fragments rather than full-length kinase

• Purification and quality enhancement:

  • Affinity purification against the recombinant protein

  • Depletion strategies using related kinase proteins

  • Validation against knockout mutant backgrounds

Research on Arabidopsis antibody development demonstrates that affinity purification significantly improves detection rates, with crude antisera often failing in immunolocalization while purified antibodies show robust signals .

How should researchers interpret contradictory results between different detection methods when using At3g51990 antibodies?

When faced with contradictory results using At3g51990 antibodies across different techniques, implement this systematic analytical approach:

• Technical assessment:

  • Evaluate epitope accessibility in different techniques

  • Consider protein conformation differences between native vs. denatured states

  • Analyze fixation/extraction method effects on epitope presentation

• Antibody characteristic analysis:

  • Determine if antibodies recognize different epitopes

  • Assess antibody affinities under varying conditions

  • Evaluate batch-to-batch variability

• Biological validation through orthogonal methods:

  • Correlation with transcript levels (qRT-PCR)

  • Verification through genetic approaches (mutant complementation)

  • Mass spectrometry validation of target protein

Studies on plant antibodies have shown that recombinant protein antibodies often perform differently in Western blot versus immunolocalization applications. For example, in comprehensive Arabidopsis antibody studies, only 22 of 38 antibodies that detected proteins were suitable for immunocytochemistry, while others worked exclusively in Western blot applications .

What refolding protocols are most effective for producing functional recombinant antibodies against At3g51990?

When producing recombinant antibodies against At3g51990, proper protein folding is critical for functionality. Implement these methodological approaches:

• Inclusion body solubilization strategy:

  • Moderate solubilization with optimized chaotropic agent concentrations

  • Controlled pH and temperature conditions during solubilization

  • Gradual denaturant removal through step-wise dialysis

• Redox environment optimization:

  • Implementation of glutathione redox pair (GSSG/GSH) at optimal ratios

  • Addition of protein disulfide isomerase to facilitate correct disulfide formation

  • Maintenance of appropriate oxidative environment

• Refolding matrix selection:

  • On-column refolding using immobilized metal affinity chromatography

  • Matrix-assisted refolding with chaperone proteins

  • Size exclusion chromatography to isolate properly folded species

Research has demonstrated that moderate-solubilization approaches combined with in vitro refolding matrices and optimal redox pairing yield more correctly folded, structured antibodies with higher functional activity compared to direct dilution methods without redox pairs .

How should researchers design experiments to analyze At3g51990 post-translational modifications using antibody-based approaches?

For investigating post-translational modifications (PTMs) of At3g51990, implement these methodological strategies:

• Modification-specific antibody development:

  • Generate phospho-specific antibodies against predicted phosphorylation sites

  • Validate specificity using phosphatase-treated samples

  • Implement peptide competition assays with phosphorylated/non-phosphorylated peptides

• Enrichment strategies for modified protein:

  • Immunoprecipitation with total protein antibodies followed by PTM detection

  • Phosphopeptide enrichment prior to mass spectrometry

  • Sequential immunoprecipitation approaches for low-abundance modifications

• Experimental design for detecting dynamic modifications:

  • Time-course analysis after stimulus application

  • Comparison between multiple tissues/developmental stages

  • Inclusion of kinase/phosphatase inhibitors to stabilize modifications

Studies on stress-responsive plant proteins demonstrate that experimental design must account for rapid PTM changes. For example, aluminum stress response studies in Arabidopsis utilized a carefully controlled experimental design with three independent replicates where control and treated samples were compared to detect stress-induced protein modifications .

What are the most effective strategies for troubleshooting non-specific binding when using At3g51990 antibodies in complex plant tissues?

When encountering non-specific binding with At3g51990 antibodies, implement this systematic troubleshooting approach:

• Blocking optimization protocol:

  • Test multiple blocking agents (BSA, milk, normal serum, plant-specific blockers)

  • Implement extended blocking times (4-16 hours at 4°C)

  • Add competing proteins from non-target species to reduce cross-reactivity

• Antibody purification enhancement:

  • Perform additional affinity purification steps

  • Implement negative selection against problematic cross-reactive proteins

  • Consider preparing F(ab')₂ fragments to reduce Fc-mediated binding

• Signal-to-noise ratio improvement:

  • Decrease primary antibody concentration while extending incubation time

  • Implement more stringent washing protocols (higher salt, longer washes)

  • Use monovalent detection systems to reduce amplification of non-specific binding

Experimental evidence from Arabidopsis antibody development shows that affinity purification dramatically improves antibody performance. For example, crude antibodies often fail in immunolocalization applications, while affinity-purified versions of the same antibodies provide specific signals .

How can researchers integrate antibody-based detection of At3g51990 with other molecular biology techniques for comprehensive functional analysis?

For comprehensive functional analysis of At3g51990, implement this integrated methodological framework:

• Multi-level expression analysis protocol:

  • Correlate protein levels (antibody detection) with transcript levels (qRT-PCR)

  • Analyze protein turnover rates using cycloheximide chase experiments

  • Combine with promoter-reporter fusions to study transcriptional regulation

• Protein interaction network mapping:

  • Co-immunoprecipitation with At3g51990 antibodies followed by mass spectrometry

  • Yeast two-hybrid screening validated by in planta methods

  • Proximity labeling approaches (BioID, APEX) with antibody validation

• Functional complementation approach:

  • Generation of mutant complementation lines expressing tagged versions

  • Phenotypic rescue analysis correlated with antibody-detected expression levels

  • Structure-function studies using domain deletions and antibody epitope mapping

Research on plant protein function shows that integration of antibody detection with genetic approaches provides the most robust functional insights. For example, in studies of Arabidopsis root proteins, antibody detection was validated through genetic approaches using corresponding mutant backgrounds .