At1g48625 Antibody

What is At1g48625 and how does it function in Arabidopsis thaliana?

At1g48625 is a gene locus in Arabidopsis thaliana that encodes a protein potentially involved in plant immune response pathways. While specific research on At1g48625 is emerging, its function may share similarities with other proteins involved in plant defense mechanisms. Based on current understanding of related proteins, it likely participates in signal transduction pathways similar to those involving NPR1 (Non-expressor of Pathogenesis-Related genes 1), which functions as a key regulator in systemic acquired resistance . The protein may interact with transcription factors to activate expression of downstream target genes involved in plant immunity, similar to how NPR1 interacts with TGA transcription factors to activate PR (Pathogenesis-Related) gene expression.

What validation methods should be employed for At1g48625 antibodies?

Validation of At1g48625 antibodies should follow a multi-step approach to ensure specificity and reliability:

• Western blot analysis: Compare protein detection in wild-type plants versus knockout/knockdown lines

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody captures the intended protein

• Immunofluorescence with controls: Include negative controls (pre-immune serum) and positive controls (tagged At1g48625 protein)

• Cross-reactivity testing: Test antibody against related proteins to assess specificity

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide to verify binding specificity

For optimal results, validation should include cell-free degradation assays similar to those used for NPR1-GFP, where protein stability and degradation rates can be assessed over defined time periods (0-180 minutes) .

How do At1g48625 antibodies compare to fluorescent protein fusion approaches?

Both approaches offer distinct advantages for protein detection:

For complete analysis, both approaches can be combined as demonstrated in studies of NPR1-GFP, where fluorescent tagging enables visualization while antibody detection confirms protein levels .

What are optimal protocols for using At1g48625 antibody in immunoprecipitation experiments?

For successful immunoprecipitation of At1g48625 protein, the following methodological approach is recommended:

• Sample preparation:

  • Harvest 2-3 g of plant tissue and grind in liquid nitrogen

  • Extract in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation (1000 × g, 5 min)

• Immunoprecipitation:

  • Add At1g48625 antibody (5-10 μg) to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 50 μl of Protein A/G beads and incubate for 3 hours

  • Wash 4× with extraction buffer

  • Elute proteins with SDS sample buffer or low pH buffer

• Analysis:

  • Perform SDS-PAGE and western blot or prepare for mass spectrometry

  • Include appropriate controls (IgG control, input sample)

Similar immunoprecipitation approaches have been effective for studying NPR1 interactions, which could serve as a methodological template .

How can At1g48625 antibody be used to study protein subcellular localization?

To determine the subcellular localization of At1g48625 protein:

• Immunofluorescence microscopy:

  • Fix plant tissues with 4% paraformaldehyde

  • Permeabilize cell walls with enzymes (cellulase, macerozyme)

  • Block with 3% BSA in PBS for 1 hour

  • Incubate with At1g48625 primary antibody (1:100-1:500 dilution)

  • Wash with PBS + 0.1% Tween-20

  • Apply fluorescent secondary antibody (1:500-1:1000)

  • Counterstain nuclei with DAPI

  • Image using confocal microscopy

• Subcellular fractionation followed by immunoblotting:

  • Isolate nuclear, cytoplasmic, and membrane fractions

  • Perform western blot with At1g48625 antibody

  • Include fraction-specific markers (e.g., histone H3 for nuclear fraction)

  • Quantify relative distribution across fractions

When analyzing nuclear versus cytoplasmic localization, consider that stimuli like plant pathogens or salicylic acid may trigger translocation between compartments, as observed with NPR1 where SA promoted nuclear translocation while still maintaining significant cytoplasmic presence .

What controls are essential when using At1g48625 antibody in western blotting?

When performing western blot analysis with At1g48625 antibody, the following controls are essential:

• Positive controls:

  • Recombinant At1g48625 protein

  • Overexpression lines of At1g48625

  • Tagged At1g48625 with known expression level

• Negative controls:

  • Knockout/knockdown lines of At1g48625

  • Pre-immune serum instead of primary antibody

  • Secondary antibody only

• Loading controls:

  • Housekeeping proteins (actin, tubulin, GAPDH)

  • Total protein stain (Ponceau S, Coomassie)

• Additional validation controls:

  • Peptide competition assay

  • Multiple antibodies targeting different epitopes of At1g48625

  • Gradient dilution series to confirm linearity of detection

Proper controls ensure reliable results and help distinguish between specific signals and artifacts, as demonstrated in studies of NPR1-GFP stability where control treatments with cycloheximide (CHX) were used to block protein synthesis and assess degradation rates .

How can At1g48625 antibody be employed in chromatin immunoprecipitation (ChIP) experiments?

For studying At1g48625 interactions with DNA using ChIP:

• Sample preparation:

  • Crosslink 3-5 g Arabidopsis tissue with 1% formaldehyde for 10 minutes

  • Quench with 0.125 M glycine

  • Extract nuclei and sonicate chromatin to 200-500 bp fragments

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads

  • Incubate with At1g48625 antibody overnight at 4°C

  • Capture antibody-protein-DNA complexes with Protein A/G beads

  • Wash extensively with increasingly stringent buffers

  • Reverse crosslinks (65°C overnight)

  • Purify DNA

• Analysis options:

  • ChIP-qPCR for known target genes

  • ChIP-seq for genome-wide binding profile

  • ChIP-reChIP to identify co-occupancy with other factors

• Data interpretation considerations:

  • Compare enrichment to input and IgG controls

  • Normalize to housekeeping gene regions

  • Identify binding motifs in enriched regions

If At1g48625 functions as a transcription factor or co-activator similar to NPR1, which interacts with transcription factors like TGAs in the nucleus to activate downstream target genes, ChIP experiments would be valuable for identifying its regulatory targets .

What techniques combine At1g48625 antibody with other molecular tools for studying protein-protein interactions?

Several advanced techniques combine antibody usage with other molecular tools:

• Co-immunoprecipitation (Co-IP):

  • Use At1g48625 antibody to pull down the protein complex

  • Identify interacting partners by mass spectrometry or immunoblotting

  • Verify results with reverse Co-IP using antibodies against potential interacting proteins

• Proximity Ligation Assay (PLA):

  • Apply At1g48625 primary antibody together with antibody against suspected interacting protein

  • Use oligonucleotide-linked secondary antibodies

  • Amplify signal when proteins are in close proximity (<40 nm)

  • Visualize discrete spots indicating interaction sites in situ

• Förster Resonance Energy Transfer (FRET) with immunolabeling:

  • Use fluorophore-conjugated At1g48625 antibody paired with differently labeled interacting protein antibody

  • Measure energy transfer as indication of protein proximity

  • Combine with fixation to capture transient interactions

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Compare antibody-based interaction results with BiFC data

  • Correlate subcellular localization patterns

These approaches could help determine if At1g48625 interacts with immune response regulators similar to how ATG6 interacts with NPR1 in both nuclear and cytoplasmic compartments .

How can At1g48625 antibody be used to study protein complex dynamics during immune response?

To study At1g48625 protein complex dynamics during immune response:

• Time-course experiments:

  • Treat plants with pathogens (e.g., Pst DC3000/avrRps4) or immune elicitors

  • Collect samples at multiple timepoints (0, 6, 12, 24 hours post-treatment)

  • Perform immunoprecipitation with At1g48625 antibody

  • Analyze complex composition changes by mass spectrometry

  • Track protein abundance changes by immunoblotting

• Subcellular translocation analysis:

  • Perform fractionation at different timepoints during immune response

  • Track At1g48625 movement between cytoplasm and nucleus using the antibody

  • Correlate with expression of immune response genes

• Protein stability assessment:

  • Perform cell-free degradation assays with At1g48625 antibody detection

  • Use cycloheximide chase experiments to determine protein half-life changes

  • Compare degradation rates between healthy and infected tissue

• Condensate formation analysis:

  • Examine if At1g48625 forms biomolecular condensates during immune response

  • Use immunofluorescence to visualize potential condensate structures

  • Determine if these structures are similar to SINCs (SA-induced NPR1 condensates)

This approach mirrors experiments showing that ATG6 expression is significantly increased after treatment with Pst DC3000/avrRps4 and SA, suggesting immune response induction .

How should researchers quantify At1g48625 protein expression across different experimental conditions?

For accurate quantification of At1g48625 protein expression:

• Western blot quantification:

  • Use standard curves with recombinant protein at known concentrations

  • Ensure detection is within linear range of antibody

  • Apply densitometry software (ImageJ, ImageLab) for band intensity measurement

  • Normalize to loading controls or total protein stain

  • Calculate relative expression or absolute quantities when standards are used

• Statistical analysis requirements:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Calculate standard deviation and standard error

  • Determine significance (p-values) of expression changes

• Visualization methods:

  • Present data as bar graphs with error bars

  • Include representative western blot images

  • Indicate statistical significance

• Controls for accurate quantification:

  • Include positive and negative controls on each blot

  • Use identical exposure times when comparing blots

  • Apply housekeeping gene normalization

This quantification approach is similar to methods used to determine that NPR1-GFP protein levels were significantly higher in ATG6-mCherry × NPR1-GFP plants compared to NPR1-GFP plants after SA treatment .

What are best practices for interpreting contradictory results when using At1g48625 antibody?

When faced with contradictory results using At1g48625 antibody:

• Antibody validation check:

  • Verify antibody specificity with western blot on wild-type vs. knockout samples

  • Test multiple antibody lots and dilutions

  • Consider epitope availability in different experimental conditions

• Experimental condition analysis:

  • Examine buffer compositions for incompatibilities

  • Check protein extraction methods for potential selective extraction

  • Verify that fixation methods preserve the epitope

  • Assess if treatments modify the protein (phosphorylation, ubiquitination)

• Alternative approaches:

  • Apply orthogonal techniques (mass spectrometry, RNA expression)

  • Use tagged protein versions alongside antibody detection

  • Try alternative antibodies targeting different epitopes

• Biological complexity considerations:

  • Assess tissue-specific or developmental stage differences

  • Consider post-translational modifications affecting antibody recognition

  • Evaluate potential isoform detection variations

When interpreting contradictory results, remember that protein behavior can change dramatically between conditions, as seen with NPR1 which shows different localization patterns and protein levels before and after SA treatment .

How can researchers address nonspecific binding when using At1g48625 antibody?

To address nonspecific binding with At1g48625 antibody:

• Optimization strategies:

  • Titrate antibody concentration to minimize background

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase washing stringency (higher salt, detergent concentration)

  • Optimize incubation times and temperatures

• Pre-adsorption techniques:

  • Pre-incubate antibody with knockout/knockdown plant extract

  • Use purified recombinant proteins of related family members

  • Apply protein extracts from non-plant species

• Analytical approaches:

  • Compare banding patterns between wild-type and knockout samples

  • Perform peptide competition assays at different concentrations

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

• Advanced purification options:

  • Affinity-purify antibody against immobilized antigen

  • Perform negative selection against common cross-reactive proteins

  • Consider monoclonal antibody development for improved specificity

Addressing nonspecific binding is critical for accurate results, particularly when studying proteins that may be part of complex networks like plant immunity pathways, where multiple related proteins might show structural similarities .

How might At1g48625 antibody contribute to understanding plant stress response pathways?

At1g48625 antibody could advance our understanding of plant stress responses through:

• Pathway mapping applications:

  • Identify At1g48625 interacting partners during biotic and abiotic stress

  • Track protein modifications (phosphorylation, ubiquitination) under stress

  • Monitor protein relocalization between subcellular compartments

  • Characterize protein complex remodeling during stress response

• Comparative biology approaches:

  • Study At1g48625 homologs across plant species with varying stress tolerance

  • Compare protein expression patterns between resistant and susceptible varieties

  • Assess conservation of protein interactions across species

• Integration with multi-omics data:

  • Correlate protein levels with transcriptome changes during stress

  • Connect proteomics and metabolomics data through At1g48625 pathways

  • Create integrated network models of stress response

• Translational research applications:

  • Develop At1g48625-based markers for stress resistance breeding

  • Screen chemical libraries for compounds that modulate At1g48625 function

  • Engineer crops with optimized At1g48625 activity

This approach mirrors the discovery that ATG6 expression is significantly upregulated after Pst DC3000/avrRps4 treatment, suggesting involvement in pathogen response pathways .

What innovative techniques might enhance At1g48625 antibody applications in the future?

Emerging techniques that could enhance At1g48625 antibody applications include:

• Single-cell proteomics:

  • Applying At1g48625 antibody for single-cell western blotting

  • Using microfluidic antibody capture for cell-specific analysis

  • Combining with single-cell transcriptomics for multi-omics integration

• Super-resolution microscopy advances:

  • STORM/PALM imaging with At1g48625 antibody for nanoscale localization

  • Expansion microscopy for improved spatial resolution of protein complexes

  • Live-cell super-resolution for dynamic protein tracking

• Antibody engineering improvements:

  • Nanobody development against At1g48625 for improved tissue penetration

  • Split-antibody complementation for interaction studies

  • Bispecific antibodies targeting At1g48625 and interacting proteins

• Spatially-resolved proteomics:

  • Combining antibody detection with laser capture microdissection

  • Imaging mass spectrometry guided by At1g48625 immunolocalization

  • Digital spatial profiling of At1g48625 alongside other proteins

These advanced approaches could help detect and characterize protein condensates similar to the SINCs-like condensates observed with NPR1, which might play important roles in immunity .

How can At1g48625 antibody research contribute to crop improvement strategies?

At1g48625 antibody research could contribute to crop improvement through:

• Disease resistance applications:

  • Identify plants with optimal At1g48625 protein levels or localization patterns

  • Screen breeding populations using antibody-based assays

  • Monitor At1g48625 protein responses to pathogens in resistant vs. susceptible lines

• Stress tolerance mechanisms:

  • Characterize At1g48625 protein behavior under drought, salt, or temperature stress

  • Correlate protein levels/modifications with stress tolerance phenotypes

  • Develop rapid screening tools for stress-responsive At1g48625 variants

• Regulatory pathway engineering:

  • Monitor effects of genetic modifications on At1g48625 and related proteins

  • Track protein network changes in improved crop varieties

  • Assess unintended consequences of breeding or engineering

• Field application development:

  • Create field-deployable antibody-based diagnostic tools

  • Develop immunochromatographic strips for rapid protein detection

  • Design high-throughput screening platforms for breeding programs

If At1g48625 functions in immunity pathways similar to ATG6 and NPR1, understanding its regulation could be valuable for developing crops with enhanced disease resistance while maintaining yield potential .