At4g05090 Antibody

What is At4g05090 and why is it important in plant research?

At4g05090 is a gene locus in Arabidopsis thaliana that encodes a protein involved in plant signaling pathways. Similar to other proteins like AT4G23050 (a PAS domain-containing protein tyrosine kinase), At4g05090 plays critical roles in plant environmental responses and developmental processes. The protein contains functional domains that participate in cellular signaling cascades, making it an important target for understanding plant adaptations to environmental stimuli . Research with antibodies targeting this protein enables scientists to investigate protein expression, localization, and functional interactions within complex plant signaling networks.

What validation techniques should be used to confirm At4g05090 antibody specificity?

Rigorous validation is essential for antibody research quality. For At4g05090 antibodies, multiple complementary approaches should be employed:

• Western blot analysis comparing wild-type and knockout/mutant plants

• Immunoprecipitation followed by mass spectrometry verification

• Blocking peptide competition assays

• Cross-species reactivity testing when applicable

• Immunofluorescence correlation with GFP-tagged protein expression

Each validation approach provides distinct evidence of specificity. For instance, in western blotting, the antibody should detect a band of the expected molecular weight in wild-type samples that is absent in knockout specimens . Additionally, peptide competition assays where the immunizing peptide blocks antibody binding provides further confirmation that the antibody specifically recognizes the intended epitope.

How should researchers interpret differences between polyclonal and monoclonal At4g05090 antibodies?

The choice between polyclonal and monoclonal antibodies significantly impacts experimental outcomes when studying At4g05090:

Researchers should consider that polyclonal antibodies against At4g05090 might provide stronger signals in applications like western blotting due to their recognition of multiple epitopes, but monoclonal antibodies offer superior consistency for longitudinal studies where reproducibility is critical . When published results show discrepancies, the antibody type may explain these differences and should be noted in experimental interpretations.

What are the optimal fixation and antigen retrieval methods for At4g05090 immunolocalization in plant tissues?

Successful immunolocalization of At4g05090 in plant tissues requires careful consideration of fixation and antigen retrieval protocols. For optimal results:

• Fixation:

  • For membrane-associated proteins like At4g05090, a combination of 4% paraformaldehyde with 0.1-0.5% glutaraldehyde preserves both protein antigenicity and structural context

  • Fixation duration should be optimized (typically 1-2 hours at room temperature or overnight at 4°C)

  • Vacuum infiltration is essential to ensure penetration into plant tissues

• Antigen retrieval:

  • Heat-mediated retrieval in citrate buffer (pH 6.0) for 10-20 minutes often improves antibody access to At4g05090 epitopes

  • Enzymatic retrieval using proteinase K (1-10 μg/ml for 5-15 minutes) can be tested if heat-mediated approaches fail

  • For cross-linked tissues, a combination of heat and detergent treatment may be necessary

These protocols should be empirically optimized for specific plant tissues, as root tissues may require different conditions than leaf tissues due to varying cell wall composition and cytoskeletal arrangements . Preliminary experiments comparing multiple fixation and retrieval methods are recommended before proceeding with full-scale studies.

How should researchers design appropriate controls for At4g05090 antibody experiments?

Robust controls are essential for reliable interpretations of At4g05090 antibody experiments:

All experimental antibody data should be evaluated against these controls to ensure scientific rigor. For instance, immunofluorescence images should always include secondary-only controls processed identically to experimental samples but omitting the primary antibody . For quantitative analyses, statistical comparisons between experimental and control samples are necessary.

What are the recommended protocols for At4g05090 protein extraction to maintain epitope integrity?

The extraction method significantly impacts antibody recognition of At4g05090 protein. Optimal protocols depend on the protein's subcellular localization and characteristics:

• Buffer composition:

  • For membrane-associated forms: Tris-HCl (50 mM, pH 7.5), NaCl (150 mM), glycerol (10%), EDTA (1 mM), and detergents (0.5-1% Triton X-100 or 0.1-0.5% SDS)

  • For cytosolic forms: Phosphate buffer (50 mM, pH 7.4), NaCl (150 mM), EDTA (1 mM)

• Protease inhibitors (essential components):

  • PMSF (1 mM)

  • Protease inhibitor cocktail with leupeptin, pepstatin, and aprotinin

  • Phosphatase inhibitors if studying phosphorylated forms

• Extraction conditions:

  • Maintain samples at 4°C throughout extraction

  • Homogenization method impacts protein yield and integrity (mortar/pestle grinding in liquid nitrogen often preferred for plant tissues)

  • Centrifugation speeds and times should be optimized for subcellular fractionation when needed

Plant-specific compounds like phenolics and polysaccharides can interfere with antibody-epitope interactions, so adding polyvinylpyrrolidone (1-2%) to extraction buffers may improve results . When comparing experimental treatments, identical extraction methods must be maintained across all samples to avoid methodology-induced artifacts.

How can At4g05090 antibodies be used to investigate protein-protein interactions in stress response networks?

At4g05090 antibodies can be strategically employed to elucidate protein interaction networks through multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use At4g05090 antibodies conjugated to beads or protein A/G to pull down the protein complex

  • Analyze interacting partners through mass spectrometry

  • Confirm interactions through reciprocal Co-IP with antibodies against putative partners

  • Application: Identifying novel protein complexes formed during stress conditions

• Proximity-dependent labeling:

  • Create fusion proteins with BioID or APEX2 systems

  • Use At4g05090 antibodies to verify fusion protein expression and localization

  • Application: Capturing transient interactions in living cells

• Bimolecular Fluorescence Complementation (BiFC):

  • Verify BiFC construct expression using At4g05090 antibodies

  • Compare immunofluorescence patterns with BiFC signals

  • Application: Visualizing interactions in specific subcellular compartments

When investigating stress responses, researchers should design experiments that capture dynamic interaction changes across a time course following stress application. For example, cold stress experiments might collect samples at 0, 15, 30, 60, and 120 minutes post-treatment to identify rapid interaction changes . Quantitative co-IP followed by western blotting with At4g05090 antibodies can reveal how interaction stoichiometry changes under different environmental conditions.

What approaches can resolve contradictory results obtained with different At4g05090 antibodies?

When researchers encounter contradictory results using different At4g05090 antibodies, a systematic approach can resolve these discrepancies:

• Epitope mapping and comparison:

  • Determine the exact epitopes recognized by each antibody

  • Assess if epitopes might be masked by protein interactions or post-translational modifications

  • Consider accessibility of epitopes in different experimental conditions

• Post-translational modification analysis:

  • Use modification-specific antibodies alongside general At4g05090 antibodies

  • Employ phosphatase or deglycosylation treatments to determine if modifications affect antibody binding

  • Consider that different antibodies may preferentially recognize modified or unmodified forms

• Methodological standardization:

  • Conduct side-by-side comparisons under identical conditions

  • Systematically vary sample preparation methods to identify protocol-dependent effects

  • Use multiple detection methods (e.g., fluorescence and chemiluminescence)

• Biological validation:

  • Use CRISPR/Cas9 edited plant lines with epitope tags

  • Compare antibody results with transcriptional data

  • Consider tissue-specific or developmental expression patterns

In one documented case study with a similar plant protein, contradictory localization results were resolved when researchers discovered that one antibody recognized a phosphorylated form predominantly in the nucleus, while another detected the unmodified cytoplasmic form . Publishing comprehensive antibody validation data alongside research findings helps the scientific community interpret seemingly contradictory results.

How can At4g05090 antibodies be applied in chromatin immunoprecipitation (ChIP) experiments to study transcriptional regulation?

For researchers investigating potential transcriptional regulatory functions of At4g05090, specialized ChIP protocols yield optimal results:

• Chromatin preparation optimization:

  • Crosslinking: 1% formaldehyde for precisely 10 minutes at room temperature

  • Quenching: 125 mM glycine for 5 minutes

  • Sonication: Optimize cycle number and power to achieve 200-500 bp fragments

  • Validation: Verify fragment size distribution by agarose gel electrophoresis

• Immunoprecipitation considerations:

  • Pre-clearing with protein A/G beads reduces background

  • Input controls (10% of chromatin before IP) are essential

  • Negative controls should include IgG from the same species as the At4g05090 antibody

  • Positive controls using antibodies against known transcription factors help validate the protocol

• Data analysis and validation:

  • qPCR for specific genomic regions of interest

  • ChIP-seq for genome-wide binding profile

  • Motif analysis to identify consensus binding sequences

  • Integration with transcriptome data to correlate binding with gene expression changes

ChIP-qPCR data should be presented as percent input or fold enrichment over IgG control, with statistical analysis comparing different experimental conditions . For reliable results, ChIP experiments should be performed with at least three biological replicates, and findings validated through orthogonal methods such as electrophoretic mobility shift assays or reporter gene assays.

What strategies can overcome weak or inconsistent signal problems with At4g05090 antibodies?

Researchers encountering signal problems with At4g05090 antibodies can implement the following systematic optimization strategies:

• Sample preparation optimization:

  • Test alternative extraction buffers that might better preserve epitope structure

  • Adjust protein denaturation conditions (temperature, reducing agents)

  • Consider native vs. denaturing conditions based on epitope characteristics

  • Verify protein transfer efficiency using reversible staining methods

• Antibody optimization:

  • Titrate antibody concentrations to determine optimal working dilution

  • Test extended incubation times (overnight at 4°C vs. 1-2 hours at room temperature)

  • Try different blocking agents (BSA, milk, specialized blocking reagents)

  • Consider antibody purification if background is problematic

• Detection system enhancement:

  • Employ signal amplification systems (e.g., biotin-streptavidin)

  • Use high-sensitivity substrates for enzymatic detection

  • Optimize exposure times for imaging

  • Consider advanced detection systems (e.g., tyramide signal amplification)

For particularly challenging applications, researchers can use recombinant protein expression systems to produce known quantities of At4g05090 protein as positive controls. This approach enables precise assessment of antibody sensitivity and can help determine the lower detection limit . Each optimization step should be systematically documented to develop reproducible protocols.

How can researchers distinguish between specific and non-specific binding in At4g05090 antibody applications?

Distinguishing specific from non-specific signals requires rigorous experimental design and validation:

When western blots show multiple bands, further investigation using genetic variants with predicted molecular weight shifts (e.g., fluorescent protein fusion constructs) can confirm which band represents the authentic target . For microscopy applications, colocalization with orthogonal markers of known subcellular compartments helps validate specific localization patterns.

What are the optimal storage and handling conditions to maintain At4g05090 antibody functionality?

Proper antibody handling significantly impacts experimental reproducibility:

• Storage recommendations:

  • Temperature: Store concentrated antibody stocks at -80°C in small aliquots to avoid freeze-thaw cycles

  • Working dilutions: Store at 4°C with preservative (0.02% sodium azide) for short term (1-2 weeks)

  • Avoid repeated freeze-thaw cycles (create single-use aliquots)

  • Protect from light, especially if conjugated to fluorophores

• Stability considerations:

  • Monitor antibody performance over time using consistent positive controls

  • Record lot numbers and purchase dates to track potential degradation

  • Consider adding protein stabilizers (BSA, glycerol) for diluted antibodies

  • Test aged antibodies against fresh lots if signal quality decreases

• Quality control practices:

  • Establish standard operating procedures for antibody handling

  • Include positive controls in every experiment to confirm antibody functionality

  • Document all handling steps and storage conditions

  • Consider antibody validation after significant changes in experimental protocols

Researchers should maintain detailed records of antibody performance over time to identify potential degradation . For critical experiments, side-by-side comparison between fresh and older antibody aliquots can help distinguish between antibody degradation and other experimental variables affecting results.

How can multiplexed detection systems be optimized for studying At4g05090 interactions with other proteins?

Advanced multiplexed detection enables simultaneous visualization of At4g05090 and its interaction partners:

• Antibody compatibility considerations:

  • Select primary antibodies from different host species to enable species-specific secondary antibodies

  • When limited by host species, use directly conjugated primary antibodies

  • Test for cross-reactivity between detection systems

  • Consider sequential detection protocols when cross-reactivity occurs

• Multiplexed imaging optimization:

  • Select fluorophores with minimal spectral overlap

  • Implement appropriate controls for bleed-through

  • Use spectral unmixing for closely overlapping signals

  • Consider signal-to-noise ratios for each channel

• Advanced multiplexing approaches:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Sequential immunofluorescence with antibody stripping

  • Proximity ligation assays for direct interaction detection

  • DNA-barcoded antibodies for highly multiplexed detection

Implementation of these techniques has enabled researchers studying similar plant proteins to visualize up to 5 different proteins simultaneously, revolutionizing our understanding of signaling complex formation during plant stress responses . When optimizing multiplexed detection, systematically test each antibody individually before combining to establish optimal conditions for each target.

What considerations are critical when using At4g05090 antibodies in quantitative proteomics workflows?

Integrating antibody-based enrichment with quantitative proteomics requires careful experimental design:

• Antibody-based enrichment strategies:

  • Immunoprecipitation followed by mass spectrometry

  • Immunoaffinity purification of protein complexes

  • Antibody-based fractionation before proteomic analysis

  • Targeted proteomic approaches using antibody enrichment

• Quantification approaches:

  • Label-free quantification: Requires highly reproducible sample preparation

  • Isotope labeling (SILAC, TMT, iTRAQ): Enables multiplexing and increased precision

  • Selected/Multiple Reaction Monitoring: Targeted approach for specific peptides

  • Data-independent acquisition: Comprehensive peptide fragmentation

• Validation requirements:

  • Confirm antibody specificity for the enrichment target

  • Use isotype controls for background assessment

  • Include spike-in standards for quantification accuracy

  • Validate key findings with orthogonal methods

When designing quantitative workflows, researchers should consider that antibody affinity may vary between different post-translationally modified forms of At4g05090 . To address this limitation, complementary enrichment strategies or modification-specific enrichment prior to immunoprecipitation can provide more comprehensive coverage of the target protein's modified forms.

How can At4g05090 antibodies contribute to understanding protein conformational changes during stress responses?

Antibodies can serve as powerful tools for detecting conformational states of At4g05090:

• Conformation-specific antibody applications:

  • Develop or identify antibodies that recognize specific conformational states

  • Use differential accessibility of epitopes to infer structural changes

  • Apply native vs. denaturing conditions to reveal conformation-dependent epitopes

  • Combine with biophysical techniques (e.g., limited proteolysis, hydrogen-deuterium exchange)

• Experimental design considerations:

  • Rapid sample collection and processing to preserve in vivo conformations

  • Comparative analysis across stress conditions and time points

  • Correlation with functional assays to link conformational changes with activity

  • Mutation analysis of key structural elements to validate conformational hypotheses

• Advanced structural biology integration:

  • Use antibodies as crystallization chaperones for structural studies

  • Apply negative-stain electron microscopy with antibody labeling

  • Implement single-molecule FRET with antibody-based fluorophore conjugation

  • Combine with molecular dynamics simulations to interpret conformational changes

For proteins like At4g05090 that may undergo significant conformational changes during signaling events, antibodies that differentially recognize active versus inactive states provide valuable research tools . By systematically characterizing epitope accessibility under different conditions, researchers can develop models of protein structural dynamics during stress response activation.