At4g10603 Antibody

What is At4g10603 and why are antibodies against it valuable in plant research?

At4g10603 encodes an S locus-related glycoprotein 1 (SLR1) binding pollen coat protein in Arabidopsis thaliana . This protein belongs to a family involved in plant reproductive biology, particularly in pollen-stigma recognition and fertilization processes. Antibodies against At4g10603 are valuable research tools because they enable:

• Visualization of protein expression patterns in different tissues

• Determination of subcellular localization

• Quantification of protein levels under different conditions

• Analysis of protein-protein interactions

• Investigation of post-translational modifications

These applications make At4g10603 antibodies essential for understanding plant reproductive mechanisms at the molecular level.

What methods are used to generate At4g10603 antibodies?

At4g10603 antibodies can be generated through several complementary approaches:

Phage display is particularly effective, allowing for in vitro selection of antibodies with high specificity from antibody libraries constructed by PCR amplification of variable heavy (VH) and variable light (VL) chains . This technique can produce antibodies with carefully selected properties for specific experimental applications.

What validation steps are essential before using At4g10603 antibodies?

Thorough validation of At4g10603 antibodies is critical before experimental use. Essential validation steps include:

• Western blot analysis using:

  • Positive control (tissue known to express At4g10603)

  • Negative control (At4g10603 knockout line)

  • Recombinant At4g10603 protein as reference

• Immunoprecipitation specificity testing:

  • Mass spectrometry confirmation of pulled-down proteins

  • Comparison with control IgG precipitation

• Immunohistochemistry controls:

  • Competing peptide blocking

  • Secondary antibody-only controls

  • Tissue-specific expression pattern verification

These validation steps ensure experimental results are truly reflective of At4g10603 biology rather than antibody artifacts.

What are the optimal Western blot conditions for At4g10603 detection?

For optimal Western blot detection of At4g10603, the following protocol parameters are recommended:

• Sample preparation:

  • Extract proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Triton X-100, 10% glycerol, with protease inhibitor cocktail

  • Sonicate briefly to disrupt plant cell walls

  • Centrifuge at 15,000×g for 10 minutes to clear debris

• Gel electrophoresis:

  • Load 20-30 μg total protein per lane

  • Use 12% SDS-PAGE for optimal separation

• Transfer conditions:

  • Transfer to PVDF membrane at 30V overnight at 4°C

  • This gentle transfer improves detection of glycoproteins like At4g10603

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in PBST (1 hour at room temperature)

  • Primary antibody dilution: 1:500 in blocking solution (overnight at 4°C)

  • Secondary antibody: HRP-conjugated anti-rabbit/mouse IgG (1:10,000 dilution)

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or image using a digital imager

  • Quantify band intensity using software like ImageJ

This optimized protocol accounts for the specific characteristics of plant proteins and provides reliable detection of At4g10603.

How can Co-immunoprecipitation with At4g10603 antibody be optimized?

Co-immunoprecipitation (Co-IP) with At4g10603 antibody requires specific optimization for plant tissues. The following protocol yields consistent results:

• Tissue selection and preparation:

  • Use reproductive tissues (anthers, pollen) for highest At4g10603 expression

  • Grind 300-500 mg tissue in liquid nitrogen

  • Extract in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Triton X-100, 10% glycerol, with protease inhibitors

• Extract clarification:

  • Centrifuge at 15,000×g for 10 minutes at 4°C

  • Pass supernatant through a 0.45 μm filter

• Pre-clearing step (critical):

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

  • Remove beads by centrifugation (800×g, 1 minute)

• Immunoprecipitation:

  • Add 5-10 μg At4g10603 antibody to pre-cleared extract

  • Incubate with gentle rotation overnight at 4°C

  • Add 50 μL pre-equilibrated Protein A/G magnetic beads

  • Incubate 3 hours at 4°C

• Washing and elution:

  • Wash beads 3 times with extraction buffer

  • Wash twice with 0.1M Tris-HCl buffer (pH 7.5)

  • Elute with 4X LDS sample buffer heated to 70°C

• Analysis:

  • Separate by SDS-PAGE

  • Identify interacting proteins by immunoblotting or mass spectrometry

This method has been adapted from successful co-IP protocols used for other plant proteins and optimized for At4g10603.

What immunohistochemistry protocol works best for At4g10603 localization?

For optimal immunohistochemistry results with At4g10603 antibody in plant reproductive tissues, the following protocol is recommended:

• Tissue fixation and embedding:

  • Fix fresh tissue in 4% paraformaldehyde in PBS (pH 7.4) for 12 hours at 4°C

  • Dehydrate through ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Clear with xylene and embed in paraffin

  • Section at 8 μm thickness

• Antigen retrieval (critical step):

  • Deparaffinize sections with xylene

  • Rehydrate through decreasing ethanol series

  • Heat in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 15 minutes

  • Cool gradually to room temperature

• Blocking and antibody incubation:

  • Block with 5% normal goat serum, 1% BSA in PBS with 0.1% Triton X-100 for 2 hours

  • Incubate with At4g10603 antibody (1:100 dilution) overnight at 4°C

  • Wash 3× with PBS (10 minutes each)

  • Incubate with fluorescent secondary antibody (1:500) for 1 hour at room temperature

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

  • Mount in anti-fade medium

  • Seal with nail polish for long-term storage

This protocol accounts for the specific challenges of plant tissues, particularly the cell wall and cuticle barriers that can impede antibody penetration.

How can At4g10603 antibody be used to study protein-protein interactions?

At4g10603 antibody enables several complementary approaches for studying protein-protein interactions in plant reproductive biology:

• Co-immunoprecipitation followed by mass spectrometry:

  • Use At4g10603 antibody to pull down protein complexes

  • Identify novel interaction partners by mass spectrometry

  • Validate findings with reciprocal co-IP using antibodies against identified partners

• Proximity ligation assay (PLA):

  • Detect protein interactions in situ with submicrometer resolution

  • Requires primary antibodies from different species against At4g10603 and potential interactors

  • Generates fluorescent signal only when proteins are in close proximity (<40 nm)

• FRET analysis with antibody fragments:

  • Label antibody fragments with appropriate FRET donor/acceptor pairs

  • Monitor real-time interactions in living cells

  • Calculate FRET efficiency to quantify interaction strength

• Validation through yeast two-hybrid:

  • Use candidate approach based on co-IP findings

  • Test direct protein-protein interactions

  • Similar to approaches described for protein interaction studies in other plant systems

These complementary approaches provide robust evidence for biologically relevant interactions involving At4g10603 protein.

How can At4g10603 antibody contribute to understanding self-incompatibility mechanisms?

At4g10603 antibody provides valuable tools for investigating self-incompatibility mechanisms in Arabidopsis and related species:

• Comparative localization studies:

  • Track At4g10603 localization during compatible versus incompatible pollinations

  • Quantify protein redistribution following pollination

  • Correlate localization patterns with pollen hydration and germination

• Phosphorylation state analysis:

  • Develop phospho-specific At4g10603 antibodies

  • Compare phosphorylation status in compatible versus incompatible reactions

  • Identify kinases involved through co-IP studies

• Protein complex dynamics:

  • Use At4g10603 antibody to isolate protein complexes at different stages of pollination

  • Identify temporal changes in interaction partners

  • Map the signaling cascade initiated during self-recognition

• Functional blocking studies:

  • Apply At4g10603 antibody fragments to stigmas before pollination

  • Assess effects on pollen recognition and acceptance

  • Identify specific domains critical for self/non-self discrimination

These approaches contribute to our understanding of the molecular mechanisms underlying self-incompatibility, with potential applications in plant breeding and reproductive biology.

What role can At4g10603 antibody play in studying CRISPR-edited plant lines?

At4g10603 antibody serves as a critical tool for analyzing CRISPR-edited plant lines, providing molecular validation of editing outcomes and functional insights:

• Validation of gene editing:

  • Confirm protein knockout in CRISPR deletion lines

  • Detect truncated proteins in frameshift mutants

  • Quantify protein reduction in knockdown lines

• Domain function analysis:

  • Create CRISPR lines with specific domain deletions

  • Use At4g10603 antibody to confirm expression of truncated proteins

  • Correlate domain presence with protein function and localization

• Tagged variant analysis:

  • Generate CRISPR knock-in lines with epitope tags

  • Compare detection using At4g10603 antibody versus tag antibodies

  • Assess whether tagging affects protein function or localization

• Rescue experiment verification:

  • Complement knockout lines with modified At4g10603 variants

  • Use antibody to confirm expression levels of complemented constructs

  • Correlate protein levels with phenotypic rescue

This integration of CRISPR technology with antibody-based detection provides powerful insights into At4g10603 structure-function relationships.

Why am I detecting multiple bands with my At4g10603 antibody?

Multiple bands in At4g10603 immunoblots can arise from several sources, each requiring specific troubleshooting approaches:

• Post-translational modifications:

  • Glycosylation heterogeneity (common in plant glycoproteins)

  • Phosphorylation states

  • Solution: Treat samples with appropriate deglycosylation enzymes or phosphatases before SDS-PAGE

• Alternative splice variants:

  • At4g10603 may have splice variants with different molecular weights

  • Solution: Compare with RNA-seq data on splice variant expression

  • Validation: Design epitope-specific antibodies to distinguish variants

• Proteolytic degradation:

  • Incomplete protease inhibition during extraction

  • Solution: Use stronger protease inhibitor cocktails

  • Prevention: Maintain strict cold-chain during sample preparation

• Cross-reactivity with related proteins:

  • At4g10603 belongs to a protein family with similar sequences

  • Solution: Pre-absorb antibody with recombinant related proteins

  • Alternative: Generate peptide antibodies against unique regions

• Non-specific binding:

  • Secondary antibody binding to endogenous plant proteins

  • Solution: Test different blocking reagents (5% milk, 2% BSA, commercial blockers)

  • Control: Include secondary antibody-only control

Systematic investigation of these possibilities will determine whether multiple bands represent biologically relevant forms or technical artifacts.

What steps should I take if I'm getting weak or no signal with At4g10603 antibody?

Weak or absent signals when using At4g10603 antibody can be addressed through systematic troubleshooting:

• Sample preparation optimization:

  • Ensure tissue selection targets sites of At4g10603 expression (pollen, anthers)

  • Optimize protein extraction buffer components

  • Concentrate proteins if expression levels are low

• Protein transfer efficiency:

  • Verify transfer success using reversible stains (Ponceau S)

  • Consider extended or modified transfer conditions for glycoproteins

  • Try different membrane types (PVDF often superior for plant proteins)

• Antibody conditions:

  • Test concentration range (1:100 to 1:2000)

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

  • Try different secondary antibodies with higher sensitivity

• Detection system sensitivity:

  • Switch to more sensitive ECL substrates

  • Consider signal amplification systems

  • Increase exposure time for film detection

• Protein expression timing:

  • At4g10603 expression may be developmental stage-specific

  • Verify developmental timing of collected tissues

  • Consider circadian or environmental factors affecting expression

This systematic approach identifies and addresses the specific limiting factor in At4g10603 detection.

How can I differentiate between specific and non-specific signals?

Distinguishing specific from non-specific signals is critical for reliable At4g10603 research. The following approaches provide definitive differentiation:

• Genetic controls:

  • Compare wild-type to At4g10603 knockout line

  • True specific signal should be absent in knockout tissue

  • Persistent bands in knockout samples represent non-specific binding

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide or recombinant protein

  • Specific signals should be blocked

  • Persistent signals despite blocking indicate non-specificity

• Multiple antibody validation:

  • Test antibodies raised against different epitopes of At4g10603

  • Specific signals should be detected by multiple antibodies

  • Signals detected by only one antibody warrant careful verification

• Correlation with expression data:

  • Compare antibody signal intensity with RNA expression patterns

  • Tissue-specific expression should correlate with protein detection

  • Discrepancies suggest potential non-specific binding

• Size validation:

  • Compare detected band size with predicted molecular weight

  • Account for post-translational modifications

  • Unexplained size discrepancies suggest non-specific detection

These validation approaches should be applied systematically to ensure experimental findings truly reflect At4g10603 biology.

How can At4g10603 antibody be used with super-resolution microscopy?

At4g10603 antibody can be adapted for super-resolution microscopy applications, providing nanoscale insights into protein distribution and interactions:

• Sample preparation considerations:

  • Fix tissues with aldehydes optimized for structure preservation

  • Use thin sections (5-8 μm) to minimize out-of-focus background

  • Consider tissue clearing techniques to improve signal-to-noise ratio

• Antibody modifications for STORM/PALM:

  • Directly conjugate At4g10603 antibody with photoswitchable fluorophores

  • Alternatively, use secondary antibodies labeled with appropriate dyes

  • Test F(ab')2 fragments for improved epitope access and reduced displacement

• Multi-color super-resolution imaging:

  • Combine At4g10603 detection with markers for subcellular structures

  • Use spectrally distinct fluorophores with minimal bleed-through

  • Include fiducial markers for drift correction and channel alignment

• Data acquisition parameters:

  • Optimize laser power to balance photoswitching and photobleaching

  • Collect 10,000-30,000 frames for comprehensive sampling

  • Use appropriate buffer systems to sustain fluorophore blinking

• Analysis approaches:

  • Cluster analysis to quantify protein organization patterns

  • Co-localization analysis at nanometer precision

  • 3D reconstruction to map protein distribution in cellular context

This cutting-edge approach reveals At4g10603 distribution at 10-25 nm resolution, far beyond the diffraction limit of conventional microscopy.

Can At4g10603 antibody be used to study evolutionary conservation across plant species?

At4g10603 antibody can be a valuable tool for evolutionary studies of pollen coat proteins across plant species:

• Cross-species reactivity testing:

  • Perform Western blots on protein extracts from related plant species

  • Test immunohistochemistry on pollen from diverse Brassicaceae

  • Create reactivity profile to map epitope conservation

• Epitope conservation analysis:

  • Correlate antibody reactivity with sequence conservation

  • Identify conserved functional domains versus divergent regions

  • Generate species-specific antibodies against divergent regions

• Functional conservation studies:

  • Compare subcellular localization patterns across species

  • Assess protein-protein interactions in different species

  • Correlate structural conservation with functional conservation

• Evolutionary adaptation investigation:

  • Compare At4g10603 expression and localization in self-compatible versus self-incompatible species

  • Examine protein modifications specific to particular evolutionary lineages

  • Link molecular changes to reproductive strategy shifts

This evolutionary perspective provides insights into the selective pressures shaping reproductive proteins and their specialized functions across plant taxa.

How can At4g10603 antibody contribute to understanding plant reproduction under stress conditions?

At4g10603 antibody enables nuanced investigation of how environmental stress affects plant reproductive mechanisms:

• Stress-induced expression changes:

  • Compare At4g10603 protein levels under various stress conditions

  • Create quantitative protein expression profiles using calibrated Western blotting

  • Correlate protein changes with reproductive success metrics

• Subcellular redistribution analysis:

  • Use immunohistochemistry to track protein localization under stress

  • Quantify changes in membrane association versus cytoplasmic distribution

  • Identify stress-specific localization patterns

• Post-translational modification shifts:

  • Develop modification-specific antibodies (phospho-specific, etc.)

  • Monitor modification states under different stress conditions

  • Link modifications to protein function or stability changes

• Stress-induced interaction network changes:

  • Perform co-immunoprecipitation under control versus stress conditions

  • Identify stress-specific interaction partners

  • Map dynamic stress-responsive protein networks

These approaches reveal how reproductive proteins respond to environmental challenges, providing insights into plant adaptation mechanisms and potentially informing breeding strategies for climate resilience.