At4g22390 Antibody

What is the At4g22390 protein and what cellular functions does it serve?

At4g22390 is a gene locus in Arabidopsis thaliana that encodes a protein involved in plant cellular processes. While not specifically detailed in the search results, antibodies against plant proteins like At4g22390 are essential tools for studying protein localization, expression patterns, and functional characterization in plant biology research. When working with plant protein antibodies, researchers should consider the protein's predicted size, localization, and regulatory mechanisms to design appropriate experimental protocols. Understanding these fundamental characteristics is crucial for interpreting experimental results and developing targeted research questions about the protein's biological function.

What are the recommended applications for At4g22390 antibodies?

Based on similar antibodies for Arabidopsis proteins, At4g22390 antibodies would likely be applicable for several experimental techniques. Western blotting (WB) is typically the primary validated application for plant protein antibodies, requiring optimization of dilution ratios (commonly starting at 1:1000) . When designing experiments, researchers should evaluate each antibody's specific validated applications as listed in technical documentation. Some antibodies may also be suitable for immunoprecipitation, immunohistochemistry, or immunofluorescence, though these applications often require separate validation studies to confirm specificity in these contexts.

What are the optimal storage and reconstitution protocols for At4g22390 antibodies?

Plant protein antibodies like those for At4g22390 typically require careful handling to maintain activity. For lyophilized antibodies, reconstitution should be performed with sterile water or buffer as specified in the product documentation. Long-term storage of lyophilized antibodies at -20°C can preserve activity for up to 3 years, while reconstituted antibodies can typically be stored at 4°C for shorter periods (days to weeks) . To prevent activity loss from repeated freeze-thaw cycles, researchers should prepare small aliquots after reconstitution. Additionally, brief centrifugation of tubes before opening is recommended to collect material that may adhere to caps or tube walls during storage and transport.

What expected molecular weight should be observed for At4g22390 in Western blot applications?

When performing Western blot analysis with At4g22390 antibodies, researchers should consult product documentation for both predicted and apparent molecular weights. For plant proteins, there is often a discrepancy between predicted molecular weight (based on amino acid sequence) and apparent molecular weight observed on SDS-PAGE due to post-translational modifications or intrinsic protein properties. For example, with similar plant antibodies, the expected and apparent molecular weights may differ significantly (e.g., 78 kDa predicted vs. 90 kDa apparent for the PHY protein) . This information is crucial for correctly identifying protein bands and interpreting experimental results.

How should optimal Western blot protocols be established for At4g22390 detection?

Establishing robust Western blot protocols for At4g22390 requires methodical optimization of several parameters. Based on protocols used for similar plant antibodies, researchers should:

• Extract total protein using buffers containing protease inhibitors (e.g., 0.1 M Tris-HCl pH 8.5, 4% SDS, 2% (v/v) 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride)

• Denature samples at 95°C for 10 minutes in Laemmli buffer

• Use 10-12% SDS-PAGE gels for optimal separation

• Transfer proteins to PVDF membranes (2 hours using semi-dry transfer)

• Block membranes with 5% milk in PBS-T (0.5% Tween) for 30 minutes at room temperature

• Incubate with primary antibody (starting at 1:1000 dilution) overnight at 4°C

• Wash thoroughly with PBS-T before secondary antibody incubation

Researchers should include appropriate controls and perform initial titration experiments to determine optimal antibody concentration for their specific experimental conditions.

What controls are essential when working with At4g22390 antibodies?

Rigorous experimental design for At4g22390 antibody applications should incorporate several critical controls:

These controls are essential for validating experimental results and troubleshooting potential issues with antibody specificity or experimental conditions.

How can expression patterns of At4g22390 be characterized under different experimental conditions?

Characterizing At4g22390 expression patterns requires systematic experimental design and careful sample preparation. Researchers should consider:

• Tissue-specific expression: Collect and process different tissues (roots, leaves, stems, flowers) separately to identify tissue-specific expression patterns.

• Developmental regulation: Sample plants at different developmental stages to track expression changes throughout the plant lifecycle.

• Environmental response: Subject plants to relevant environmental conditions (light/dark cycles, temperature stress, drought, pathogen exposure) and collect samples at defined timepoints.

• Quantitative analysis: Implement densitometry analysis of Western blot results for semi-quantitative comparison across conditions, normalizing to appropriate loading controls.

• Complementary approaches: Validate protein expression patterns with transcript analysis (qRT-PCR) to distinguish transcriptional from post-transcriptional regulation .

This systematic approach allows researchers to build a comprehensive understanding of At4g22390 regulation under diverse physiological conditions.

What strategies can verify antibody specificity for At4g22390?

Confirming antibody specificity is critical for research validity. For At4g22390 antibodies, researchers should implement multiple verification strategies:

• Genetic approaches: Compare protein detection in wild-type versus knockout/knockdown mutants (e.g., T-DNA insertion lines like SALK_056328C) . Absence of signal in knockout lines strongly supports antibody specificity.

• Peptide competition assays: Pre-incubate antibody with excess immunizing peptide before application to samples. Specific binding should be blocked, while non-specific binding will remain.

• Heterologous expression: Express recombinant At4g22390 with epitope tags in systems like E. coli and confirm detection with both tag-specific and At4g22390-specific antibodies.

• Multiple antibody validation: If available, compare results using antibodies raised against different epitopes of At4g22390.

• Mass spectrometry confirmation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein.

Documentation of these specificity tests significantly strengthens research findings and should be included in publications.

How can inconsistent Western blot results with At4g22390 antibodies be addressed?

Troubleshooting inconsistent results requires systematic evaluation of each experimental step:

• Sample preparation issues:

  • Ensure complete protein extraction with fresh protease inhibitors

  • Verify protein integrity by Coomassie staining of duplicate gels

  • Standardize protein quantification methods

• Antibody-related factors:

  • Test different antibody dilutions (1:500 to 1:2000)

  • Prepare fresh antibody dilutions from stock

  • Extend primary antibody incubation time (overnight at 4°C)

  • Verify antibody storage conditions and expiration dates

• Detection protocol optimization:

  • Adjust blocking conditions (duration, blocking agent)

  • Optimize wash steps (duration, buffer composition)

  • Evaluate alternative secondary antibodies

  • Adjust detection system sensitivity

• Biological variables:

  • Control growth conditions precisely between experiments

  • Standardize harvesting times to account for circadian regulation

  • Consider plant developmental stage variations

Keeping detailed experimental records helps identify patterns in variability and facilitates targeted optimization.

What approaches can resolve cross-reactivity issues with At4g22390 antibodies?

Managing cross-reactivity challenges requires both experimental and analytical approaches:

• Experimental modifications:

  • Increase antibody dilution to reduce non-specific binding

  • Optimize blocking conditions (try different blocking agents: BSA, casein, commercial blockers)

  • Increase wash stringency (duration, detergent concentration)

  • Consider alternative antibody options (monoclonal vs. polyclonal)

• Sample preparation refinements:

  • Implement additional purification steps (subcellular fractionation)

  • Pre-clear lysates with appropriate beads/matrices before antibody application

  • Adjust detergent type/concentration in extraction buffers

• Analytical validation:

  • Compare observed bands with predicted molecular weights of potential cross-reactive proteins

  • Confirm protein identity through mass spectrometry

  • Validate results with genetic approaches (mutant lines)

  • Test antibody reactivity against predicted cross-reactive species (based on sequence homology)

Documenting cross-reactivity profiles enhances experimental transparency and facilitates protocol optimization across the research community.

How can immunoprecipitation protocols be optimized for At4g22390 studies?

Immunoprecipitation (IP) optimization for plant proteins like At4g22390 requires careful consideration of multiple factors:

• Extraction buffer composition:

  • Test different detergent types and concentrations (NP-40, Triton X-100, CHAPS)

  • Adjust salt concentration to balance extraction efficiency with preservation of protein-protein interactions

  • Include appropriate protease and phosphatase inhibitors

• Antibody coupling methods:

  • Compare direct coupling to beads versus indirect capture with Protein A/G

  • Determine optimal antibody concentration for IP

  • Test cross-linking options to prevent antibody leaching

• IP conditions:

  • Optimize incubation temperature and duration

  • Determine appropriate washing stringency

  • Develop elution protocols that maximize target recovery

• Validation approaches:

  • Confirm protein identity through Western blotting and mass spectrometry

  • Include appropriate controls (IgG control, input samples)

  • Validate with complementary approaches (e.g., reciprocal IP with interacting proteins)

Researchers should develop custom protocols based on At4g22390's specific properties and interactions, rather than relying solely on generic IP procedures.

How can generative AI approaches support At4g22390 antibody research?

Recent developments in generative artificial intelligence offer promising applications for antibody research:

• Antibody design optimization:
  Generative AI models can predict optimal antibody sequences for specific targets like At4g22390, potentially improving specificity and affinity. These approaches leverage deep learning to model antibody-antigen interactions and design complementarity-determining regions (CDRs) with enhanced binding properties .

• Epitope prediction and optimization:
  AI models can analyze the At4g22390 protein sequence to identify immunogenic epitopes that are likely to generate high-quality antibodies, improving immunization strategy design .

• Cross-reactivity assessment:
  Predictive models can evaluate potential cross-reactivity with related plant proteins, helping researchers select epitopes that maximize specificity for At4g22390 over homologous proteins .

• Structure-based antibody development:
  When combined with protein structure prediction (e.g., AlphaFold), generative AI approaches can design antibodies optimized for specific structural features of At4g22390 .

While these technologies are still emerging, they represent promising avenues for developing next-generation research antibodies with enhanced performance characteristics.

What methods can characterize post-translational modifications of At4g22390?

Comprehensive characterization of At4g22390 post-translational modifications (PTMs) requires integrating multiple experimental approaches:

• Specialized antibody applications:

  • Modification-specific antibodies (phospho, acetyl, ubiquitin, SUMO)

  • Two-dimensional Western blotting to separate modified forms

  • Phos-tag gels for phosphorylation detection

• Mass spectrometry approaches:

  • Immunoprecipitation followed by MS analysis

  • Enrichment strategies for specific modifications

  • Label-free or isotope-labeling quantification

• Functional validation:

  • Site-directed mutagenesis of modified residues

  • In vitro modification assays

  • Pharmacological inhibition of modifying enzymes

• Subcellular localization impacts:

  • Correlation between modifications and protein localization

  • Dynamics of modification in response to stimuli

  • Co-localization with modifying enzymes

These approaches provide complementary information about At4g22390 regulation and function, enabling researchers to develop mechanistic models of protein activity in different cellular contexts.

How can At4g22390 antibodies contribute to plant systems biology research?

At4g22390 antibody research can significantly advance plant systems biology through:

• Protein interaction network mapping:

  • Immunoprecipitation coupled with mass spectrometry to identify At4g22390 interactors

  • Proximity labeling approaches (BioID, APEX) to identify spatial protein networks

  • Validation of predicted protein-protein interactions from computational models

• Multi-omics integration:

  • Correlation of protein abundance data with transcriptomics

  • Integration with metabolomic profiles to link At4g22390 function to metabolic pathways

  • Combining with phosphoproteomics to understand signaling networks

• Dynamic response profiling:

  • Tracking At4g22390 abundance and modification state across environmental conditions

  • Temporal resolution of protein regulation during stress responses

  • Developmental trajectory mapping

• Functional module characterization:

  • Defining At4g22390's position within functional protein complexes

  • Determining regulatory relationships within biochemical pathways

  • Establishing hierarchical organization of signaling cascades

These approaches contribute to comprehensive understanding of plant cellular systems and regulatory networks, providing insights that extend beyond individual protein characterization.

What considerations apply when developing custom At4g22390 antibodies?

Researchers developing custom antibodies against At4g22390 should consider:

• Epitope selection criteria:

  • Analyze protein sequence for regions with high antigenicity and surface accessibility

  • Avoid regions with high sequence conservation across protein families to minimize cross-reactivity

  • Consider epitopes that span functionally important domains for potential blocking antibodies

  • Select peptide length appropriate for the application (typically 10-20 amino acids)

• Host species selection:

  • Choose host species based on phylogenetic distance from plants

  • Consider intended applications (rabbit polyclonal antibodies offer versatility; monoclonal antibodies provide consistency)

  • Plan for secondary antibody compatibility in multi-labeling experiments

• Validation requirements:

  • Design validation experiments using genetic controls (knockouts/knockdowns)

  • Prepare for cross-reactivity testing against related proteins

  • Establish application-specific validation protocols (Western blot, IHC, IP)

• Production considerations:

  • For KLH-conjugated peptides, specify conjugation chemistry

  • Consider affinity purification against the immunizing peptide

  • Request detailed immunization and purification protocols from providers

Thorough planning and comprehensive validation ensure that custom antibodies become valuable, reliable research tools.