At1g56220 Antibody

What is At1g56220 and why is it important to study with antibodies?

At1g56220 encodes a dormancy/auxin associated family protein in Arabidopsis thaliana . This protein is significant in plant biology research because it plays a role in auxin translocation at the plasma membrane and is involved in phosphorylation-dependent signaling pathways . Antibodies against At1g56220 allow researchers to investigate its expression patterns, subcellular localization, and potential functional roles in plant development and stress responses. The protein's involvement in auxin signaling makes it particularly relevant for understanding fundamental aspects of plant growth regulation and environmental adaptation mechanisms.

What types of antibodies are available for At1g56220 protein detection?

Researchers can utilize both conventional antibodies and recombinant antibodies for At1g56220 detection:

• Conventional polyclonal antibodies: Typically raised against synthetic peptides or purified recombinant At1g56220 protein fragments

• Monoclonal antibodies: Provide higher specificity but may be less available for plant-specific proteins

• Recombinant antibodies: Offer significant advantages including unambiguous identification through DNA sequencing, reliable expression, and ease of distribution as DNA sequences or plasmids

For plant proteins like At1g56220, many researchers prefer recombinant antibody technology as it allows for engineering modifications to enhance utility and reproducibility in plant tissue applications .

How should I design experiments to validate an At1g56220 antibody for specificity?

A comprehensive validation approach should include:

• Western blot analysis with appropriate controls:

  • Wild-type Arabidopsis tissue lysates

  • Knockout/knockdown mutant samples (if available)

  • Overexpression lines expressing tagged At1g56220

  • Peptide competition assays

• Immunoprecipitation followed by mass spectrometry:

  • This can confirm antibody specificity by identifying pulled-down proteins

  • Follow protocols similar to those used in phosphoproteome analyses

• Immunolocalization experiments:

  • Compare results with fluorescent protein fusion localization patterns

  • Include appropriate negative controls

  • Use confocal microscopy to determine subcellular localization

• Cross-reactivity testing:

  • Test against closely related dormancy/auxin associated family proteins

  • Evaluate potential cross-reactivity with proteins in other plant species if using the antibody across species

What expression systems are recommended for producing recombinant At1g56220 antibodies?

When producing recombinant antibodies against At1g56220, consider using plant-based expression systems for applications requiring plant-specific post-translational modifications, particularly if the antibody will be used for in vivo studies .

What are the optimal protocols for using At1g56220 antibodies in immunoprecipitation experiments?

For effective immunoprecipitation of At1g56220:

• Tissue preparation and lysis buffer optimization:

  • Use young tissue with higher protein expression

  • Optimal lysis buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% SDS, protease inhibitor cocktail, and phosphatase inhibitors

  • For phosphorylation studies, include phosphatase inhibitors like those used in phosphoproteome analyses: "100 mM TEAB (triethyl ammonium bicarbonate), and cOmplete Ultra Protease Inhibitor Cocktail and PhosSTOP"

• Antibody binding conditions:

  • Pre-clear lysate with protein A/G beads

  • Use 2-5 μg antibody per 500 μg total protein

  • Incubate overnight at 4°C with gentle rotation

• Washing and elution:

  • Perform 4-5 washes with decreasing salt concentration

  • Elute with either low pH buffer or by boiling in SDS sample buffer

• Verification:

  • Western blot a portion of the immunoprecipitated material

  • Consider mass spectrometry analysis to confirm identity and potential interacting partners

How can At1g56220 antibodies be used to investigate protein-protein interactions in auxin signaling pathways?

To investigate At1g56220 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use At1g56220 antibody to pull down the protein complex

  • Analyze precipitated proteins by mass spectrometry or western blot

  • Compare results under different conditions (e.g., with/without auxin treatment)

• Proximity labeling methods:

  • Create fusion constructs of At1g56220 with BioID or APEX2

  • Use the antibody to validate expression of the fusion protein

  • Identify proximal proteins after biotin labeling

• Bimolecular Fluorescence Complementation (BiFC):

  • Validate BiFC results with antibody staining

  • Confirm protein expression levels of split fluorescent protein fusions

• Yeast two-hybrid validation:

  • Use antibodies to confirm expression levels of bait and prey proteins

  • Correlate interaction strength with protein expression levels

This approach can help identify components of auxin signaling networks and determine if At1g56220 interacts with known auxin transport or response proteins .

What are common causes of non-specific binding when using At1g56220 antibodies and how can they be addressed?

Common causes of non-specific binding and their solutions:

For improved specificity when working with At1g56220, consider using engineered recombinant antibodies with enhanced target binding affinity and/or target selectivity .

How can I address low signal issues when detecting At1g56220 in plant tissues?

To improve At1g56220 detection in challenging plant samples:

• Optimize protein extraction:

  • Use extraction buffers containing 150 mM NaCl, 100 mM TEAB with protease inhibitors

  • Consider using sonication (37°C in a water bath sonicator for 30 min) to improve extraction efficiency

  • For membrane-associated proteins like At1g56220, include 1% SDS or other appropriate detergents

• Signal amplification strategies:

  • Use biotin-streptavidin detection systems

  • Apply tyramide signal amplification for immunohistochemistry

  • Consider proximity ligation assay (PLA) for detecting low abundance proteins

• Sample enrichment:

  • Perform subcellular fractionation to concentrate plasma membrane fractions

  • Use phosphopeptide enrichment techniques if studying phosphorylated forms

  • Consider immunoprecipitation before western blotting

• Antibody optimization:

  • Try different antibody concentrations and incubation conditions

  • Consider using recombinant antibody formats with enhanced binding properties

  • Use signal-enhancing secondary antibodies or direct labeling

How can At1g56220 antibodies be used to investigate its role in SR45-mediated alternative splicing?

Research findings indicate that the sr45-1 mutation causes dramatic alterations in the splicing pattern of At1g56220 . To investigate this relationship:

• RNA-protein interaction studies:

  • Perform RNA immunoprecipitation (RIP) using At1g56220 antibodies

  • Cross-link RNA-protein complexes before immunoprecipitation

  • Analyze pulled-down RNA by RT-PCR or sequencing

• Splicing pattern analysis:

  • Use At1g56220 antibodies to detect different protein isoforms resulting from alternative splicing

  • Compare protein expression patterns between wild-type and sr45-1 mutant plants

  • Correlate protein isoform abundance with transcript isoforms

• Functional validation:

  • Create constructs expressing specific At1g56220 splicing variants

  • Use antibodies to confirm expression levels

  • Analyze phenotypes and relate to SR45 function in sugar and ABA signaling

• Mechanistic studies:

  • Investigate if SR45 directly binds to At1g56220 pre-mRNA

  • Determine if At1g56220 protein interacts with SR45 or splicing machinery components

  • Explore feedback regulation between protein levels and splicing patterns

What approaches can be used to study At1g56220 phosphorylation dynamics using phospho-specific antibodies?

Phosphorylation of At1g56220 may be critical for its function in auxin translocation . To study these dynamics:

• Development of phospho-specific antibodies:

  • Generate antibodies against predicted or known phosphorylation sites

  • Validate specificity using phosphatase treatments and phosphomimetic mutants

  • Test cross-reactivity with non-phosphorylated forms

• Temporal dynamics of phosphorylation:

  • Monitor phosphorylation changes after auxin treatment at different time points

  • Compare with total At1g56220 levels using standard antibodies

  • Correlate phosphorylation with physiological responses

• Spatial distribution analysis:

  • Use phospho-specific antibodies in immunofluorescence to determine subcellular localization of phosphorylated At1g56220

  • Compare with total protein distribution

  • Investigate co-localization with known auxin transporters or signaling components

• Identification of regulatory kinases/phosphatases:

  • Use phospho-antibodies to monitor At1g56220 phosphorylation after treatment with kinase/phosphatase inhibitors

  • Screen for potential regulatory enzymes using candidate approaches

  • Validate interactions using co-immunoprecipitation and in vitro assays

How can At1g56220 antibodies be combined with CRISPR-Cas9 gene editing for functional studies?

A comprehensive approach combining antibodies with CRISPR-Cas9 techniques:

• Validation of gene editing efficiency:

  • Use At1g56220 antibodies to confirm protein knockout or reduction in CRISPR-edited lines

  • Quantify protein levels in partial knockouts or specific domain deletions

  • Validate specificity of editing by assessing related family members

• Protein domain function analysis:

  • Generate CRISPR-edited plants with specific domain mutations or deletions

  • Use antibodies to confirm stable expression of modified proteins

  • Investigate changes in protein localization, interaction, or post-translational modifications

• Complementation studies:

  • Reintroduce modified versions of At1g56220 into knockout backgrounds

  • Use antibodies to confirm expression levels match native conditions

  • Correlate protein levels with phenotypic rescue

• Advanced editing applications:

  • Create epitope-tagged endogenous At1g56220 using CRISPR knock-in strategies

  • Compare detection using At1g56220 antibodies versus tag antibodies

  • Develop CRISPR activation/inhibition systems and monitor effects on At1g56220 expression

What strategies can be used to investigate At1g56220's role in nitrogen assimilation and storage pathways?

Since At1g56220 appears in the context of auxin signaling, which interacts with nitrogen metabolism in plants , researchers can:

• Expression analysis under varying nitrogen conditions:

  • Use At1g56220 antibodies to quantify protein levels under different N treatments

  • Compare with transcript levels to identify post-transcriptional regulation

  • Correlate with nitrogen assimilation markers

• Protein-protein interaction studies:

  • Investigate interactions between At1g56220 and known nitrogen-responsive master regulatory genes (CCA1, GLK1, bZIP11)

  • Use antibodies in co-immunoprecipitation experiments under varying N conditions

  • Validate interactions with BiFC or FRET, confirmed by antibody detection

• Subcellular localization changes:

  • Monitor At1g56220 localization changes in response to nitrogen availability

  • Correlate with auxin distribution patterns

  • Investigate co-localization with nitrogen transport or assimilation proteins

• Functional studies in N-metabolism mutants:

  • Analyze At1g56220 expression and phosphorylation in nitrogen assimilation mutants

  • Use antibodies to determine if protein stability or modification is affected

  • Investigate if At1g56220 affects expression of nitrogen-responsive genes

How can multi-omics approaches incorporate At1g56220 antibody-based studies?

Integrating antibody-based research into multi-omics frameworks:

• Integration with transcriptomics:

  • Correlate At1g56220 protein levels (detected by antibodies) with transcript abundance

  • Investigate post-transcriptional regulation mechanisms

  • Study protein-RNA interactions using techniques like RIP-seq with At1g56220 antibodies

• Proteomics integration:

  • Use antibodies to validate mass spectrometry-based proteomics findings

  • Employ immunoprecipitation followed by mass spectrometry (IP-MS) to identify protein complexes

  • Compare protein abundance across different experimental conditions or tissues

• Metabolomics connections:

  • Correlate At1g56220 protein levels or modifications with metabolite profiles

  • Investigate if At1g56220 affects auxin metabolite distributions

  • Study metabolic changes in plants with altered At1g56220 expression

• Epigenomic studies:

  • Investigate if At1g56220 interacts with chromatin components using antibody-based approaches

  • Explore potential roles in transcriptional regulation, particularly in auxin-responsive or dormancy-related genes

  • Consider connections to histone methylation, given the context of SDG8 studies

What are the considerations for developing At1g56220 antibodies compatible with super-resolution microscopy techniques?

For super-resolution microscopy applications:

• Antibody format selection:

  • Smaller formats like Fab fragments and nanobodies provide better resolution due to decreased "linkage error"

  • Single-domain antibodies (nanobodies) offer distinct advantages with their ~2-3nm size

  • Consider site-specific fluorophore conjugation rather than secondary antibody detection

• Fluorophore considerations:

  • Choose photostable fluorophores compatible with specific super-resolution techniques

  • For STORM/PALM: photoswitchable fluorophores

  • For STED: fluorophores resistant to high-intensity depletion laser

• Sample preparation optimization:

  • Develop fixation protocols that preserve antigen without introducing autofluorescence

  • Consider expansion microscopy compatible antibodies

  • Test antibody performance after various clearing techniques

• Validation approaches:

  • Compare antibody labeling with fluorescent protein fusions at conventional resolution first

  • Perform quantitative assessment of labeling density and specificity

  • Use appropriate controls including knockout lines and peptide competition