At1g10090 Antibody

What is the At1g10090 protein and why are antibodies against it valuable for plant reproduction research?

At1g10090 encodes an unknown protein in Arabidopsis thaliana with expression detected in both sporophytic and gametophytic tissues . The protein has been identified as a sperm cell-expressed gene that is being analyzed to confirm its function in reproductive processes . Antibodies against this protein are valuable for:

• Detecting native protein expression patterns to validate GFP fusion studies

• Immunoprecipitating protein complexes to identify interaction partners

• Studying subcellular localization in reproductive tissues

• Analyzing protein expression levels across developmental stages

Its expression in sperm cells makes it particularly interesting for plant reproductive biology studies, as very few sperm cell-specific proteins have been well-characterized.

What expression pattern has been established for At1g10090 in plant tissues?

At1g10090 shows expression in multiple tissues with particularly notable expression in reproductive structures:

• Expression has been detected in both sporophytic (vegetative) and gametophytic (reproductive) tissues

• The gene has been identified as a sperm cell-expressed gene

• GFP translational fusion studies have confirmed its expression pattern

• At1g10090 is classified as specifically expressed ("S") rather than preferentially expressed ("P")

This expression pattern suggests a potential specialized role in plant reproduction, particularly in male gametophyte function.

How should researchers choose between polyclonal and monoclonal antibodies for At1g10090 detection?

For At1g10090, polyclonal antibodies might be preferable for initial characterization due to the limited knowledge about this protein and its potentially low expression levels .

How can researchers validate the specificity of an At1g10090 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For At1g10090 antibodies, a comprehensive validation strategy should include:

• Western blot analysis: Test against wild-type Arabidopsis tissues alongside negative controls (At1g10090 knockout mutants)

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide before immunodetection to confirm epitope specificity

• Immunostaining comparison: Compare antibody staining patterns with At1g10090::GFP localization patterns

• Mass spectrometry verification: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein

• Cross-reactivity assessment: Test antibody against closely related proteins or in heterologous expression systems

Researchers should be particularly careful with validation since At1g10090 encodes an unknown protein with limited characterization .

What are the challenges in detecting low-abundance proteins like At1g10090 in plant reproductive tissues?

Detecting low-abundance proteins in reproductive tissues presents several challenges:

• Limited tissue availability: Male gametophytic tissues, particularly sperm cells, are extremely small and difficult to isolate in sufficient quantities

• High background interference: Plant reproductive tissues often contain high levels of autofluorescent compounds and secondary metabolites

• Protein extraction difficulties: Specialized tissues may require optimized extraction protocols to maintain protein integrity

• Signal-to-noise ratio concerns: The relatively low expression levels of At1g10090 (indicated by the "S|34|0|Spec|17" classification) may make detection challenging against background signals

• Temporal expression dynamics: Expression may be restricted to specific developmental stages, necessitating precise sampling

Researchers can address these challenges through techniques like fluorescence-activated cell sorting (FACS) for sperm cell isolation, as demonstrated for similar studies of sperm-expressed genes .

How can At1g10090 antibodies facilitate the study of protein-protein interactions in plant reproduction?

At1g10090 antibodies can be powerful tools for identifying protein interaction networks in reproductive processes:

• Co-immunoprecipitation (Co-IP): Pull down At1g10090 protein complexes from plant reproductive tissues to identify interaction partners, followed by:

  • Western blot analysis for known candidates

  • Mass spectrometry for unbiased identification

  • Targeted proteomics for low-abundance interactors

• Proximity-dependent labeling: Combine antibodies with techniques like BioID or APEX to identify proximal proteins in living cells

• In situ protein-protein interaction detection:

  • Proximity ligation assay (PLA) using At1g10090 antibody and antibodies against candidate interactors

  • Fluorescence resonance energy transfer (FRET) with labeled antibodies

• Validation through reciprocal Co-IP: Confirm interactions by immunoprecipitating with antibodies against identified partners

These approaches can help elucidate the functional role of At1g10090 in plant reproduction, potentially identifying novel components of sperm cell function or fertilization mechanisms .

What is the optimal protocol for western blot analysis using At1g10090 antibodies?

Optimized Western Blot Protocol for At1g10090 Detection:

• Sample preparation:

  • Extract proteins from reproductive tissues using buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1mM EDTA, and plant protease inhibitor cocktail

  • For sperm cells, consider specialized isolation techniques like FACS

  • Use 4-6M urea in extraction buffer if protein is membrane-associated

• Gel electrophoresis and transfer:

  • Load 50-100μg total protein per lane (higher amounts for reproductive tissues)

  • Use 10-12% polyacrylamide gels for optimal resolution

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

• Immunodetection:

  • Block membrane in 5% non-fat milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature

  • Incubate with primary anti-At1g10090 antibody (1:500 to 1:1000 dilution) overnight at 4°C

  • Wash 4 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000 dilution) for 1 hour at room temperature

  • Develop using ECL substrate and detect using a chemiluminescence imager

• Controls:

  • Include At1g10090 knockout plant extracts as negative controls

  • Use recombinant At1g10090 protein or overexpression lines as positive controls

  • Include loading controls such as anti-actin antibody (ACT3)

How can immunohistochemistry be optimized for At1g10090 detection in plant reproductive tissues?

Optimized Immunohistochemistry Protocol for At1g10090 in Reproductive Tissues:

• Tissue fixation and processing:

  • Fix fresh tissues in 4% paraformaldehyde in PBS for 2-4 hours at room temperature

  • For pollen/sperm cells, collect on poly-L-lysine coated slides and fix for 30 minutes

  • Dehydrate and embed in paraffin or prepare for cryosectioning (preferred for preserving epitopes)

  • Section at 8-10μm thickness

• Antigen retrieval and blocking:

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95°C for 10 minutes

  • Block endogenous peroxidases with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal goat serum, 1% BSA, and 0.3% Triton X-100 in PBS for 2 hours

• Antibody incubation:

  • Incubate with primary anti-At1g10090 antibody (1:100 to 1:200 dilution) in blocking buffer overnight at 4°C

  • Wash 3 times with PBS containing 0.1% Tween-20

  • Incubate with fluorescently-labeled secondary antibody (1:300) for 2 hours at room temperature

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

• Imaging considerations:

  • Use confocal microscopy for high-resolution imaging of reproductive structures

  • Acquire z-stacks to capture three-dimensional information

  • Include appropriate filter sets to distinguish antibody signal from plant autofluorescence

  • Compare staining patterns with At1g10090::GFP localization data

What approaches can be used to enhance detection sensitivity for low-abundance proteins like At1g10090?

Several approaches can enhance detection sensitivity for low-abundance proteins:

For At1g10090, combining FACS-based sperm cell isolation with signal amplification techniques would likely provide optimal sensitivity.

How can researchers address weak or absent signals when using At1g10090 antibodies?

When facing weak or absent signals, consider the following troubleshooting strategies:

• Protein extraction optimization:

  • Try alternative extraction buffers (RIPA, urea-based, or specialized plant extraction buffers)

  • Add protease inhibitors to prevent degradation

  • Include reducing agents (DTT or β-mercaptoethanol) at appropriate concentrations

  • Consider non-denaturing conditions if the epitope is conformation-dependent

• Sample preparation adjustments:

  • Increase protein loading (up to 100-150μg per lane)

  • Enrich for target protein through fractionation or concentration

  • For tissues with known expression, like sperm cells, use enrichment techniques

• Antibody optimization:

  • Titrate antibody concentration (try 1:100 to 1:1000 range)

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

  • Test different antibody lots or sources

  • Consider alternative antibodies targeting different epitopes

• Detection enhancement:

  • Use more sensitive detection substrates like ECL Plus or Femto substrates

  • Extend exposure times during imaging

  • Try signal amplification systems (biotin-streptavidin)

  • Optimize image acquisition settings on detection instruments

• Control experiments:

  • Verify antibody function with positive controls (recombinant protein)

  • Confirm protein transfer efficiency with reversible membrane staining

  • Test antibody in tissues with confirmed expression via GFP fusion studies

What strategies can minimize non-specific binding and background in At1g10090 antibody applications?

To minimize non-specific binding and background:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, casein, commercial blockers)

  • Increase blocking time (2-3 hours) and concentration (5-10%)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

  • For plant tissues, include 1-2% polyvinylpyrrolidone (PVP) to absorb phenolics

• Antibody preparation:

  • Pre-absorb antibody with plant extracts from knockout lines

  • Use affinity-purified antibody fractions

  • Dilute antibody in fresh blocking buffer

  • Centrifuge diluted antibody before use to remove aggregates

• Wash optimization:

  • Increase wash buffer volumes (use at least 10x membrane volume)

  • Extend wash times (5 washes of 5-10 minutes each)

  • Add higher detergent concentrations in wash buffers

  • Include salt (up to 500mM NaCl) to reduce ionic interactions

• Detection adjustments:

  • Reduce secondary antibody concentration (1:10,000 to 1:20,000)

  • Minimize exposure time during imaging

  • Use secondary antibodies with minimal cross-reactivity to plant proteins

  • Consider alternate detection methods with lower background

• Sample-specific considerations:

  • For plant reproductive tissues, include antioxidants in buffers

  • Use protease inhibitors to prevent artifacts from proteolysis

  • Test multiple fixation protocols for immunohistochemistry

How should researchers interpret discrepancies between At1g10090::GFP localization and antibody staining patterns?

When facing discrepancies between At1g10090::GFP fusion studies and antibody staining :

• Evaluate protein tagging effects:

  • GFP fusion may alter protein localization, stability, or function

  • The fusion might disrupt interaction with cellular trafficking machinery

  • Compare N-terminal and C-terminal GFP fusions to identify potential artifacts

• Consider epitope accessibility:

  • The antibody epitope may be masked in certain cellular contexts

  • Protein-protein interactions could block antibody binding sites

  • Post-translational modifications might affect epitope recognition

  • Try different fixation and permeabilization methods to improve accessibility

• Assess technical differences:

  • GFP visualization is performed in living cells while immunostaining requires fixation

  • Chemical fixatives can alter protein localization or epitope conformation

  • The GFP tag might be cleaved in vivo, resulting in free GFP signals

  • Antibody specificity might be insufficient to distinguish closely related proteins

• Perform validation experiments:

  • Use subcellular fractionation followed by western blotting

  • Apply super-resolution microscopy for more precise localization

  • Perform immuno-electron microscopy for ultrastructural localization

  • Compare results across different developmental stages and tissue types

• Resolve through complementary approaches:

  • Use multiple antibodies targeting different epitopes

  • Perform reciprocal experiments with tagged and untagged proteins

  • Include appropriate controls (knockout lines, preimmune serum)

  • Consider that both methods might reveal different aspects of the protein's biology

How can At1g10090 antibodies contribute to understanding plant reproductive biology?

At1g10090 antibodies can advance plant reproductive biology research through multiple applications:

• Developmental expression analysis:

  • Track protein expression throughout reproductive development

  • Correlate protein levels with specific developmental events

  • Compare expression patterns between wild-type and mutant backgrounds

  • Analyze post-translational modifications during reproduction

• Functional characterization:

  • Identify protein interaction partners in reproductive tissues

  • Map subcellular localization during fertilization processes

  • Perform chromatin immunoprecipitation if DNA-binding functions are suspected

  • Analyze protein dynamics during pollen tube growth and fertilization

• Mutant phenotype analysis:

  • Compare protein expression in wild-type and reproductive mutants

  • Investigate protein mislocalization in plants with fertility defects

  • Study compensation mechanisms in knockout/knockdown lines

  • Correlate protein levels with fertility phenotypes in T-DNA insertion lines

• Evolutionary studies:

  • Compare protein expression and localization across plant species

  • Investigate conservation of function in reproductive processes

  • Analyze species-specific differences in protein regulation

As At1g10090 has been identified as a sperm cell-expressed gene , antibodies against this protein are particularly valuable for studying male gametophyte development and function.

What considerations are important when designing experiments to correlate At1g10090 expression with fertility phenotypes?

When correlating At1g10090 expression with fertility phenotypes:

• Experimental design considerations:

  • Include appropriate controls (wild-type, known fertility mutants)

  • Use multiple independent T-DNA insertion lines

  • Perform reciprocal crosses to distinguish maternal and paternal effects

  • Design time-course experiments to capture dynamic expression changes

• Phenotypic analysis approaches:

  • Quantify seed set as a measure of fertility

  • Assess pollen viability using multiple methods (Alexander staining, FDA)

  • Analyze pollen tube growth in vitro and in vivo

  • Examine fertilization rates through aniline blue staining

  • Quantify transmission efficiency of mutant alleles

• Protein expression analysis:

  • Compare protein levels across developmental stages

  • Correlate expression with specific cellular events during reproduction

  • Analyze protein modifications that might regulate activity

  • Examine protein localization changes in response to reproductive cues

• Data integration strategies:

  • Correlate transcript levels (RT-PCR) with protein abundance (western blot)

  • Integrate expression data with phenotypic measurements

  • Use statistical approaches to establish significant correlations

  • Consider multifactorial analyses to account for environmental variables

• Validation approaches:

  • Perform complementation studies with the wild-type gene

  • Create rescue constructs with tissue-specific promoters

  • Use inducible expression systems to control timing of expression

  • Combine with CRISPR/Cas9 editing for precise genetic manipulation

How can researchers integrate At1g10090 antibody data with other molecular approaches for comprehensive protein characterization?

Integrating antibody-based data with other approaches provides comprehensive protein characterization:

For At1g10090, researchers have already integrated GFP fusion studies with RT-PCR analysis and T-DNA insertion screening , creating a foundation for further characterization using antibody-based approaches.