At2g30890 Antibody

What is the At2g30890 protein and what is its significance in Arabidopsis thaliana?

At2g30890 (UniProt ID: O80854) is a protein encoded by a gene located on chromosome 2 of Arabidopsis thaliana, commonly known as mouse-ear cress. This protein belongs to the diverse proteome of this model plant organism. Understanding this protein's function contributes to our knowledge of plant molecular biology and potentially agricultural applications. Researchers typically investigate its function through a combination of genomic, transcriptomic, and proteomic approaches, with antibody-based detection being a crucial method for studying its expression patterns and interactions .

How are antibodies against plant proteins like At2g30890 typically generated?

Antibodies against plant proteins such as At2g30890 are typically generated through several approaches:

• Recombinant protein expression: The target protein or a fragment is expressed in bacterial, insect, or mammalian expression systems, purified, and used for immunization.

• Synthetic peptide approach: Short peptide sequences (typically 10-20 amino acids) unique to At2g30890 are synthesized, conjugated to carrier proteins like KLH (keyhole limpet hemocyanin), and used for immunization.

• Immunization protocols: These typically involve multiple injections into host animals (commonly rabbits for polyclonal antibodies or mice for monoclonal antibodies) over several weeks to months.

• Screening and purification: The resulting antibodies are screened for specificity using techniques like ELISA, Western blotting, and immunoprecipitation, followed by affinity purification against the antigen to enhance specificity .
  The choice between polyclonal and monoclonal antibodies depends on the research application, with polyclonal antibodies offering broader epitope recognition and monoclonal antibodies providing higher specificity for a single epitope.

What validation documentation should accompany a reliable At2g30890 antibody?

Comprehensive validation documentation is essential for antibody reliability. For At2g30890 antibody, researchers should expect:

• Application-specific validation data: Evidence demonstrating performance in specific applications like Western blotting, immunoprecipitation, or immunohistochemistry.

• Validation methodology details: Documentation of the validation methods used, such as orthogonal validation, genetic knockdown, recombinant expression, independent antibodies, and/or capture mass spectrometry analysis.

• Species reactivity information: Clear indication of which species the antibody recognizes, particularly important for plant proteins that may have homologs across species.

• Positive and negative controls: Data showing reactivity in samples known to express At2g30890 and lack of reactivity in samples without the target.

• Lot-specific performance data: Information about consistency between production lots, as antibody performance can vary between batches .
  The Human Protein Atlas approach of providing all primary validation data on their website represents an excellent model for antibody documentation, ensuring transparency and reproducibility in research applications.

How can I validate the specificity of At2g30890 antibody for my experimental system?

Validating antibody specificity is crucial for reliable research results. For At2g30890 antibody, implement multiple validation strategies:

• Orthogonal validation: Compare protein expression levels detected by the antibody with antibody-independent methods like mass spectrometry or RNA sequencing. A correlation coefficient above 0.5 between antibody-based detection and orthogonal methods indicates good validation .

• Genetic validation: Use gene-specific siRNA or CRISPR-Cas9 to knock down At2g30890 expression in your experimental system. A specific antibody will show reduced signal intensity proportional to the knockdown efficiency .

• Recombinant expression validation: Overexpress the At2g30890 protein in a system with low endogenous expression. The antibody should detect increased signal at the appropriate molecular weight.

• Independent antibody validation: Compare results using two antibodies targeting different epitopes of At2g30890. Concordant results from both antibodies provide strong evidence of specificity .

• Capture mass spectrometry: Cut out the band recognized by the antibody in a Western blot and perform mass spectrometry to confirm the presence of At2g30890 peptides within the band .
  Implementation of at least two different validation methods is recommended to ensure high confidence in antibody specificity.

What controls should I include when using At2g30890 antibody in Western blot experiments?

Rigorous controls are essential for interpreting Western blot results with At2g30890 antibody:

• Positive control: Include a sample known to express At2g30890, such as specific Arabidopsis tissues or cell lines where the protein has been previously detected.

• Negative control: Use samples from tissues or experimental conditions where At2g30890 is not expressed, or ideally, from At2g30890 knockout lines if available.

• Loading control: Include detection of a housekeeping protein (e.g., actin, tubulin, or GAPDH) to normalize for variations in protein loading and transfer efficiency.

• Primary antibody controls:

  • Omit primary antibody incubation to assess secondary antibody non-specific binding

  • If available, use pre-immune serum from the same animal used to generate the antibody

  • Include blocking peptide competition where the primary antibody is pre-incubated with excess target peptide to demonstrate binding specificity

• Molecular weight markers: Always include standards to verify that the detected band matches the expected molecular weight of At2g30890 .
  The presence of multiple bands should be critically evaluated, as they may represent different isoforms, post-translational modifications, degradation products, or non-specific binding.

How do I troubleshoot unexpected banding patterns when using At2g30890 antibody?

When encountering unexpected banding patterns with At2g30890 antibody, systematic troubleshooting is required:

• Multiple bands:

  • Verify if alternate splicing variants or post-translational modifications of At2g30890 exist

  • Optimize blocking conditions (try different blockers like BSA, milk, or commercial blocking buffers)

  • Increase washing stringency (longer washes, higher detergent concentration)

  • Titrate antibody concentration to find optimal dilution

  • Try different detection systems with varying sensitivity

• No bands or weak signal:

  • Confirm protein extraction efficiency from plant tissues

  • Verify protein transfer efficiency (use reversible staining like Ponceau S)

  • Try different antigen retrieval methods if applicable

  • Increase antibody concentration or incubation time

  • Use more sensitive detection methods (enhanced chemiluminescence or fluorescent detection)

• High background:

  • Increase blocking time or blocker concentration

  • Reduce primary and secondary antibody concentrations

  • Add non-ionic detergents (0.05-0.1% Tween-20) to washing and antibody dilution buffers

  • Ensure membrane is never allowed to dry during the procedure .
    For persistent issues, validation using orthogonal methods or genetic approaches becomes especially important to confirm specificity.

What are the best practices for optimizing immunoprecipitation with At2g30890 antibody?

Optimizing immunoprecipitation (IP) with At2g30890 antibody requires attention to several critical parameters:

• Lysis buffer optimization:

  • Test different lysis buffers (RIPA, NP-40, Triton X-100) to find the optimal buffer that preserves At2g30890 protein structure while effectively disrupting cellular components

  • Consider adding protease inhibitors, phosphatase inhibitors, and EDTA to prevent protein degradation

  • Optimize salt concentration to balance between maintaining protein-protein interactions and reducing non-specific binding

• Antibody coupling strategies:

  • Direct approach: Covalently couple At2g30890 antibody to activated beads (such as CNBr-activated Sepharose or commercial coupling kits)

  • Indirect approach: Use Protein A/G beads to capture the antibody-antigen complex

  • Pre-clear lysates with beads alone to reduce non-specific binding

• IP conditions optimization:

  • Titrate antibody-to-lysate ratio to find optimal concentration

  • Optimize incubation time and temperature (typically 2-16 hours at 4°C)

  • Determine optimal washing stringency that removes non-specific binding without disrupting specific interactions

• Elution method selection:

  • Denaturing conditions (SDS buffer with heating) for downstream Western blot analysis

  • Native conditions (peptide competition, pH change) for activity assays or interaction studies

• Validation controls:

  • Perform IP with non-relevant antibody of the same isotype

  • Include no-antibody control

  • If studying protein interactions, validate with reciprocal IP when possible .
    For co-immunoprecipitation studies specifically targeting At2g30890 protein interactions, chemical crosslinking prior to cell lysis may help preserve transient or weak interactions.

How can I effectively use At2g30890 antibody for immunohistochemistry in plant tissues?

Effective immunohistochemistry (IHC) with At2g30890 antibody in plant tissues requires specialized approaches:

• Tissue fixation and processing:

  • Test different fixatives (paraformaldehyde, glutaraldehyde, or combinations)

  • Optimize fixation time to balance antigen preservation and tissue morphology

  • Consider using vacuum infiltration to ensure fixative penetration in plant tissues

  • Use appropriate embedding media (paraffin, resin, or cryosectioning) based on the antigen sensitivity

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (citrate or EDTA buffer)

  • Enzymatic retrieval (proteinase K, trypsin)

  • Test multiple methods as At2g30890 epitopes may respond differently

• Blocking and antibody incubation:

  • Use plant-specific blocking agents to reduce background (normal serum plus 1-3% BSA)

  • Include plant powder in blocking solution to absorb antibodies that might cross-react with plant components

  • Optimize antibody dilution and incubation time (typically 1:50-1:500, overnight at 4°C)

• Detection systems:

  • For brightfield microscopy: HRP-based systems with DAB substrate

  • For fluorescence: Select fluorophores with spectral properties distinct from plant autofluorescence

  • Consider signal amplification methods (tyramide signal amplification) for low-abundance proteins

• Controls:

  • Include sections from At2g30890 knockout or knockdown plants if available

  • Perform peptide competition controls

  • Include no-primary antibody controls .
    When optimizing protocols, begin with tissues known to have high expression of At2g30890 based on transcriptomic data to establish working conditions before examining tissues with potentially lower expression.

What methods are recommended for quantifying At2g30890 protein expression levels?

Accurate quantification of At2g30890 protein expression requires careful methodology selection and implementation:

What is the recommended sample preparation protocol for detecting At2g30890 in Arabidopsis tissues?

Effective sample preparation for At2g30890 detection requires careful consideration of tissue-specific characteristics:

• Tissue collection and storage:

  • Harvest tissues at consistent developmental stages and time of day to control for circadian expression patterns

  • Flash-freeze collected tissues in liquid nitrogen immediately after harvesting

  • Store samples at -80°C to preserve protein integrity

  • Avoid repeated freeze-thaw cycles that can lead to protein degradation

• Protein extraction methods:

  • For general extraction: Grind tissue to fine powder in liquid nitrogen, then extract in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • For membrane proteins: Include 0.1% SDS or specialized detergents like digitonin or n-dodecyl-β-D-maltoside

  • For nuclear proteins: Use specialized nuclear extraction protocols with nuclear lysis buffers

• Sample clarification:

  • Centrifuge extracts at 14,000 × g for 15 minutes at 4°C

  • For tough tissues, consider additional clarification steps such as filtration through miracloth

• Protein quantification:

  • Use Bradford, BCA, or similar assays compatible with your extraction buffer

  • Adjust all samples to equal protein concentration

• Sample denaturation:

  • Add Laemmli buffer and heat at 95°C for 5 minutes for standard SDS-PAGE

  • For membrane proteins, heating at 70°C for 10 minutes may better preserve protein structure

  • In some cases, avoid boiling completely if the protein is heat-sensitive .
    For tissues with high levels of interfering compounds (phenolics, polysaccharides), consider incorporating polyvinylpolypyrrolidone (PVPP) in the extraction buffer or using TCA/acetone precipitation to purify proteins before analysis.

How can I develop a multiplexed assay incorporating At2g30890 antibody with other targets?

Developing multiplexed assays with At2g30890 antibody enables simultaneous analysis of multiple proteins:

• Western blot multiplexing:

  • Use antibodies from different host species (e.g., rabbit anti-At2g30890 with mouse anti-reference protein)

  • Employ fluorescently-labeled secondary antibodies with distinct emission spectra

  • Ensure primary antibodies have been validated for multiplexing to avoid cross-reactivity

  • If using chemiluminescence, sequential detection with stripping between antibodies may be necessary

• Immunofluorescence multiplexing:

  • Select primary antibodies from different host species

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Include appropriate controls to assess bleed-through between channels

  • Consider sequential detection for closely related targets

• Multiplex immunoprecipitation:

  • Use antibody cocktails for co-immunoprecipitation of multiple targets

  • Validate that antibodies don't interfere with each other's binding

  • Analyze precipitated proteins using multiplex Western blot or mass spectrometry

• Optimization considerations:

  • Titrate each antibody individually before combining

  • Verify that detection sensitivity isn't compromised in multiplex format

  • Ensure reference proteins don't mask detection of At2g30890 due to abundance differences .
    For complex plant samples, consider incorporating subcellular fractionation before multiplexed analysis to reduce sample complexity and improve detection of low-abundance proteins.

What approaches can confirm the reliability of At2g30890 antibody across different experimental conditions?

Ensuring At2g30890 antibody reliability across different experimental conditions requires systematic validation:

• Batch-to-batch consistency testing:

  • Maintain reference samples (positive controls) to test each new antibody lot

  • Document performance metrics (sensitivity, specificity, background) for comparison

  • Consider creating a laboratory reference standard of purified At2g30890 protein

• Cross-application validation:

  • Validate the antibody independently for each application (Western blot, IP, IHC)

  • Don't assume performance in one application predicts performance in another

  • Document optimal conditions for each application

• Environmental variable testing:

  • Assess antibody performance across relevant temperature ranges

  • Test buffer system variations (pH, ionic strength)

  • Evaluate stability after freeze-thaw cycles

• Validation across plant growth conditions:

  • Test antibody performance in plants grown under different light conditions, temperatures, or stress treatments

  • Verify specificity in plants at different developmental stages

  • Compare performance in different plant tissues (leaves, roots, flowers, etc.)

• Documentation and standardization:

  • Create detailed protocols specifying validated conditions

  • Maintain a laboratory database of antibody performance

  • Include comprehensive validation data in publications .
    For collaborative projects, exchange validation samples between laboratories to ensure consistent antibody performance across different experimental settings and operator techniques.

What emerging technologies might enhance At2g30890 antibody applications in plant research?

The landscape of antibody applications continues to evolve with promising technologies that may enhance At2g30890 research:

• Single-cell proteomics integration:

  • Combining antibody-based detection with single-cell isolation techniques

  • Adapting cell-specific antibody labeling for plant protoplasts

  • Developing microfluidic platforms for high-throughput single-cell protein analysis

• Advanced imaging technologies:

  • Super-resolution microscopy for nanoscale localization of At2g30890

  • Expansion microscopy adapted for plant cells

  • Live-cell imaging using minimally disruptive antibody fragments

• Antibody engineering advancements:

  • Development of recombinant antibodies with enhanced specificity

  • Creation of nanobodies (single-domain antibodies) for improved tissue penetration

  • CRISPR-based epitope tagging for endogenous protein detection

• High-throughput validation platforms:

  • Automated antibody validation pipelines incorporating multiple pillars of validation

  • Standardized reporting frameworks for antibody performance metrics

  • Community-based validation resources specific to plant research .

• Computational approaches:

  • Machine learning algorithms for predicting optimal antibody applications

  • Integration of antibody-based data with multi-omics datasets

  • Improved epitope prediction for rational antibody design
    These emerging technologies offer exciting possibilities for expanding our understanding of At2g30890's role in plant biology through increasingly precise and comprehensive methodological approaches.

How can I contribute to improving antibody standards in the plant research community?

Individual researchers can significantly contribute to improving antibody standards:

• Rigorous validation reporting:

  • Publish detailed validation data for At2g30890 antibody applications

  • Include all negative results and limitations in publications

  • Share protocols with specific conditions for optimal performance

• Data repositories and sharing:

  • Submit validation data to community repositories

  • Share reagents through material transfer agreements or commercial channels

  • Participate in collaborative validation initiatives

• Method standardization:

  • Adopt the five pillars of antibody validation in your research

  • Implement minimum reporting standards for antibody experiments

  • Use consistent nomenclature and terminology across publications

• Education and training:

  • Train students and colleagues in proper antibody validation techniques

  • Organize workshops or webinars on antibody best practices

  • Serve as a reviewer advocating for proper antibody documentation

• Commercial engagement:

  • Provide detailed feedback to antibody vendors about performance

  • Participate in beta testing of new antibody products

  • Advocate for comprehensive validation data with commercial products . By implementing these practices, researchers working with At2g30890 antibody can contribute to a more robust and reproducible foundation for plant biology research.