At2g25305 Antibody

What is At2g25305 and why is it studied in plant research?

At2g25305 encodes a defensin-like (DEFL) family protein in Arabidopsis thaliana (Mouse-ear cress) . This protein belongs to a family involved in plant defense mechanisms, making it significant for studies on plant immunity, pathogen responses, and cellular signaling pathways. The protein appears in WRKY75 target lists, suggesting it may be regulated by this transcription factor and potentially involved in stress response pathways . Understanding its expression and function provides valuable insights into plant defense mechanisms and potential applications in crop protection strategies.

What applications are validated for At2g25305 antibodies?

At2g25305 antibodies have been validated for several key research applications:

Always perform validation in your experimental system as reactivity may vary between antibody lots and sample preparation methods.

What are the optimal storage conditions for At2g25305 antibodies?

For maximum stability and activity retention, store At2g25305 antibodies at -20°C or -80°C upon receipt . Avoid repeated freeze-thaw cycles by preparing working aliquots. The antibody is typically provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When working with the antibody, always keep it on ice, and centrifuge briefly before opening to collect any material adhering to the cap or sides.

How should I design Western blot experiments using At2g25305 antibody?

When designing Western blot experiments with At2g25305 antibody, follow these research-optimized protocols:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 10% glycerol, 150mM NaCl, 0.1% NP-40, 1mM PMSF, and 1× protease inhibitor cocktail

  • Load 30-50μg total protein per lane

  • Include appropriate controls (wild-type, knockout, overexpression lines)

• Electrophoresis and transfer:

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

  • Transfer to PVDF or nitrocellulose membrane (PVDF may provide better results for plant proteins)

  • Verify transfer efficiency with reversible protein stain

• Immunodetection:

  • Block with 3-5% BSA or 5% non-fat milk in TBS-T

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

  • Wash extensively with TBS-T

  • Use HRP-conjugated secondary antibody at 1:10,000 dilution

  • Develop using enhanced chemiluminescence

The expected molecular weight for At2g25305 is derived from its amino acid sequence, though post-translational modifications may affect migration patterns.

How can I determine antibody specificity when working with At2g25305?

Confirming antibody specificity is critical for reliable research outcomes. Multiple complementary approaches are recommended:

• Genetic controls:

  • Compare wild-type samples with At2g25305 knockout/knockdown lines

  • Use overexpression lines as positive controls

  • If possible, test in heterologous expression systems

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific bands should disappear in the peptide-blocked sample

• Multiple detection methods:

  • Confirm Western blot results with ELISA or other techniques

  • Correlate protein detection with mRNA expression data

  • Consider mass spectrometry validation of detected bands

• Cross-reactivity assessment:

  • Test antibody against related DEFL family proteins

  • Evaluate in non-target species (not reactive in Brassica napus, Populus sp., Rosa chinensis, Triticum aestivum per similar antibody patterns)

Document all specificity controls in your research publications to establish data reliability.

What are the optimal sample preparation methods for detecting At2g25305 in different plant tissues?

Effective sample preparation varies by tissue type and developmental stage:

For all tissues, use fresh material when possible or flash-freeze in liquid nitrogen and store at -80°C. Pulverize frozen tissue thoroughly before adding extraction buffer at a 1:3 (w/v) ratio. Include phosphatase inhibitors if studying phosphorylation states.

How can At2g25305 antibody be used to investigate protein-protein interactions?

For investigating protein-protein interactions involving At2g25305, consider these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Crosslink proteins in vivo using formaldehyde (1%, 10 min)

  • Extract proteins under gentle conditions to preserve complexes

  • Immunoprecipitate with At2g25305 antibody conjugated to protein A/G beads

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

• Proximity labeling:

  • Generate fusion proteins with BioID or APEX2 proximity labeling enzymes

  • Express in Arabidopsis using appropriate promoters

  • Identify neighboring proteins through biotinylation

  • Confirm interactions using At2g25305 antibody

• FRET-based approaches:

  • Combine At2g25305 antibody with fluorescently labeled secondary antibodies

  • Use fluorescently tagged candidate interacting proteins

  • Measure FRET signals in fixed cells or tissues

Remember that protein complexes can be dynamic and context-dependent, so experimental conditions should mimic physiological states relevant to your research question.

How does At2g25305 expression change under different stress conditions?

Understanding expression patterns under different stresses requires combining protein detection with transcriptional analysis:

When designing stress experiments:

• Include appropriate time points (early: 0-6h, intermediate: 12-24h, late: 48-72h)

• Monitor both transcript and protein levels in parallel

• Consider post-translational modifications that may affect function without changing total protein levels

• Use At2g25305 antibody in combination with subcellular fractionation to detect potential relocalization

What are the technical considerations for immunolocalization of At2g25305 in plant tissues?

Successful immunolocalization requires attention to several technical factors:

• Fixation protocols:

  • For paraffin sections: 4% paraformaldehyde in PBS, 12h at 4°C

  • For cryosections: 2% paraformaldehyde with 0.1% glutaraldehyde, 2h at room temperature

  • Carefully optimize fixation time to preserve antigenicity while maintaining structure

• Antigen retrieval:

  • Heat-mediated: 10mM sodium citrate buffer (pH 6.0), 95°C for 10-20 min

  • Enzymatic: Proteinase K (1-5 μg/ml) for 5-15 min at 37°C

  • Test multiple methods as defensin-like proteins may require specific approaches

• Antibody incubation:

  • Primary antibody: 1:200-1:500 dilution, overnight at 4°C

  • Secondary antibody: fluorophore-conjugated, 1:500 dilution, 2h at room temperature

  • Include negative controls (primary antibody omission, pre-immune serum)

• Signal detection:

  • Fluorescence microscopy offers superior resolution and colocalization capability

  • Confocal microscopy recommended for subcellular localization

  • Consider spectral imaging if autofluorescence is problematic

For subcellular localization, correlate immunostaining with bioinformatic predictions of localization signals and GFP-fusion protein studies when available.

Why might Western blots with At2g25305 antibody show multiple bands or high background?

Multiple bands or high background can arise from several experimental factors:

• Multiple bands:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splicing variants

  • Proteolytic degradation during sample preparation

  • Cross-reactivity with related defensin-like family proteins

Resolution approaches:

• Include phosphatase treatment controls if phosphorylation is suspected

• Optimize protease inhibitor cocktail composition

• Compare with transcript analysis data to identify potential isoforms

• Perform peptide competition assays to identify specific vs. non-specific bands

• High background:

  • Insufficient blocking

  • Excessive antibody concentration

  • Protein overloading

  • Incompatible membrane or detection system

Resolution approaches:

• Increase blocking time/concentration (5% BSA often works better than milk for plant samples)

• Titrate antibody concentration (try 1:2000-1:5000 dilutions)

• Add 0.05-0.1% Tween-20 to antibody dilution buffer

• Increase washing duration and number of washes

How can I optimize At2g25305 antibody for low-abundance samples?

When working with tissues where At2g25305 is expressed at low levels:

• Sample enrichment:

  • Perform immunoprecipitation before Western blot

  • Fractionate samples to concentrate relevant cellular compartments

  • Consider using plant tissues/conditions where expression is highest

• Signal amplification:

  • Use high-sensitivity chemiluminescent substrates

  • Employ tyramide signal amplification systems

  • Consider biotin-streptavidin detection systems

• Technical optimization:

  • Increase antibody incubation time (overnight at 4°C)

  • Reduce washing stringency slightly

  • Use PVDF membrane instead of nitrocellulose

  • Optimize transfer conditions for proteins in the expected MW range

• Quantitative approaches:

  • Consider ELISA as an alternative to Western blot for higher sensitivity

  • Digital droplet PCR for transcript detection may complement protein data

  • Mass spectrometry-based targeted proteomics for absolute quantification

What are the critical quality control steps for ensuring reproducible results with At2g25305 antibody?

Maintaining reproducibility requires systematic quality control:

• Antibody validation:

  • Test each new lot against a reference sample

  • Document key characteristics (detection limit, optimal dilution)

  • Store validation data and blot images for long-term reference

• Experimental standardization:

  • Maintain consistent sample preparation protocols

  • Use the same protein quantification method consistently

  • Include internal loading controls (housekeeping proteins)

  • Prepare master mixes for critical reagents

• Technical consistency:

  • Standardize exposure times for chemiluminescence detection

  • Use digital imaging systems with linear dynamic range

  • Apply quantitative analysis using software with background correction

  • Normalize target bands to loading controls

• Documentation practices:

  • Record all experimental parameters in a laboratory information system

  • Include comprehensive methods sections in publications

  • Consider sharing raw blot images in supplementary materials

  • Document any deviations from standard protocols

How can At2g25305 antibody be used in functional genomics studies?

At2g25305 antibody provides valuable capabilities for functional genomics:

• Protein expression correlation:

  • Compare protein levels with transcriptomic data across tissues or conditions

  • Identify post-transcriptional regulation mechanisms

  • Examine protein stability under different conditions

• Genetic modification studies:

  • Validate knockout/knockdown efficiency at protein level

  • Analyze overexpression lines quantitatively

  • Examine compensatory changes in related proteins

• Protein-DNA interaction studies:

  • Use in ChIP experiments if At2g25305 has DNA-binding capability

  • Identify target genes and regulatory elements

  • Map genomic binding sites through ChIP-seq

• Regulatory network analysis:

  • Combine with studies of WRKY75 and other transcription factors

  • Map signaling cascades through phosphorylation state analysis

  • Identify co-regulated proteins through parallel detection

What methodological approaches combine At2g25305 antibody with other techniques for comprehensive protein characterization?

Advanced research requires integrative approaches:

• Multi-omics integration:

  • Correlate Western blot data with RNA-seq transcriptomics

  • Combine with metabolomics to examine functional consequences

  • Integrate with phosphoproteomics for signaling studies

• Advanced microscopy:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging with complementary fluorescent protein fusions

  • Correlative light and electron microscopy for ultrastructural context

• Protein structure-function studies:

  • Epitope mapping to understand antibody binding sites

  • Combine with recombinant protein expression and purification

  • Support structural biology approaches (X-ray crystallography, cryo-EM)

• Single-cell approaches:

  • Adaptation for flow cytometry of protoplasts

  • Single-cell Western techniques for cell-to-cell variability

  • Spatial transcriptomics with protein detection

How might At2g25305 research contribute to broader understanding of plant defense mechanisms?

The strategic research implications extend to several important areas:

• Evolutionary conservation:

  • Compare At2g25305 with defensin-like proteins across species

  • Examine structural and functional conservation

  • Investigate selection pressures on defensin diversity

• Signaling network integration:

  • Map interactions with known defense pathways

  • Identify regulatory connections to WRKY transcription factors

  • Determine position in signaling cascades through temporal studies

• Biotechnological applications:

  • Evaluate potential for engineering enhanced pathogen resistance

  • Develop reporter systems based on At2g25305 promoter activity

  • Design synthetic defense networks incorporating defensin-like proteins

• Translational research:

  • Explore homologs in crop species for agricultural applications

  • Investigate potential antimicrobial properties

  • Consider applications in plant protection strategies