At2g11200 Antibody

What is AT2G11200 and why are antibodies against it important in plant research?

AT2G11200 is an F-box family protein in Arabidopsis thaliana that plays a role in protein-protein interactions and ubiquitin-mediated protein degradation pathways. This 154 amino acid protein (17671.60 Da) is predicted to primarily localize to the nucleus with a SUBAcon score of 0.651 . The protein contains F-box domains (cyclin-like and Skp2-like) that are critical for its function in cellular signaling and regulation.

Antibodies against AT2G11200 are valuable research tools for:

• Studying protein expression patterns across different tissues

• Investigating protein localization through immunohistochemistry

• Analyzing protein-protein interactions involving this F-box protein

• Examining protein abundance changes under different experimental conditions

• Validating gene knockout or knockdown experiments

Understanding this protein's role requires specific antibodies that can detect it with high specificity, particularly when investigating plant developmental pathways and stress responses.

What are the key considerations when selecting an antibody against AT2G11200?

When selecting an antibody against AT2G11200, researchers should consider:

• Antibody Specificity: Ensure the antibody recognizes AT2G11200 without cross-reactivity to other F-box proteins, particularly its closest homolog (F-box and associated interaction domains-containing protein, AT3G17320.1) .

• Antibody Format: Determine whether polyclonal or monoclonal antibodies are more suitable for your application. Monoclonal antibodies offer higher specificity to a single epitope, while polyclonal antibodies recognize multiple epitopes and may provide stronger signals .

• Species Reactivity: Confirm whether the antibody is specific to Arabidopsis thaliana or if it cross-reacts with homologous proteins in other plant species, which could be advantageous for comparative studies .

• Validated Applications: Verify that the antibody has been validated for your specific application (Western blot, immunohistochemistry, ELISA, etc.) with supporting data .

• Epitope Information: Understanding which region of AT2G11200 the antibody recognizes is critical, especially when working with protein variants or studying protein interactions where epitope accessibility may be affected.

• Purification Method: Consider whether the antibody was purified using affinity methods like Protein G, which can affect its performance in certain applications .

How can I optimize Western blot protocols for AT2G11200 detection?

Optimizing Western blot protocols for AT2G11200 detection involves several critical considerations:

• Sample Preparation:

  • Extract proteins using a buffer containing protease inhibitors to prevent degradation of AT2G11200

  • Consider nuclear enrichment protocols since AT2G11200 is primarily localized to the nucleus

  • Use fresh tissue whenever possible, as F-box proteins can be unstable

• Gel Selection and Running Conditions:

  • Use 12-15% polyacrylamide gels for optimal resolution of this ~17.7 kDa protein

  • Consider specialized techniques such as Phos-tag polyacrylamide gel electrophoresis if investigating potential phosphorylation states

• Transfer and Blocking:

  • Optimize transfer time (typically 60-90 minutes at 100V) for this relatively small protein

  • Use 5% non-fat dry milk or BSA in TBST for blocking (test both to determine which gives lower background)

• Antibody Incubation:

  • Start with a concentration of 1-2 μg/mL (similar to recommendations for plant actin antibodies)

  • Optimize incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

  • Include proper controls, including a blocking peptide control if available

• Detection and Visualization:

  • Consider enhanced chemiluminescence (ECL) detection for standard applications

  • For quantitative analysis, fluorescent secondary antibodies may provide better linearity

• Validation Controls:

  • Include positive controls (tissues known to express AT2G11200)

  • Include negative controls (knockout lines or tissues with minimal expression)

What immunoprecipitation strategies work best for studying AT2G11200 protein interactions?

Effective immunoprecipitation (IP) of AT2G11200 requires careful methodological considerations:

• Lysate Preparation:

  • Use a lysis buffer that maintains protein-protein interactions while effectively solubilizing membrane-associated proteins (typically containing 0.5-1% NP-40 or Triton X-100)

  • Include both protease and phosphatase inhibitors to preserve native interactions

  • Consider crosslinking with formaldehyde (0.1-1%) before lysis for capturing transient interactions

• Pre-clearing Strategy:

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

  • Pre-incubate with a non-specific antibody of the same isotype to absorb proteins that bind non-specifically to antibodies

• Antibody Coupling:

  • Covalently couple antibodies to beads using dimethyl pimelimidate (DMP) to prevent antibody leaching into the eluted sample

  • Alternatively, use commercially available magnetic beads pre-coated with protein G

• IP Controls:

  • Include an IgG control from the same species as the AT2G11200 antibody

  • Include a sample from AT2G11200 knockout plants as a negative control

• Elution Strategies:

  • For mass spectrometry analysis: elute with Laemmli buffer without reducing agents, add reducing agents later

  • For functional studies: consider gentle elution with competing peptides

• Verification Methods:

  • Confirm IP success by Western blot using a portion of the IP sample

  • Consider a reciprocal IP using antibodies against suspected interaction partners

This approach is particularly valuable for identifying components of SCF (Skp1-Cullin-F-box) complexes that may contain AT2G11200 as their F-box component.

Why might I observe multiple bands when probing for AT2G11200 in Western blots?

Observing multiple bands when detecting AT2G11200 can occur for several biological and technical reasons:

To address these issues:

• Optimize sample preparation:

  • Use stronger denaturing conditions if complexes are suspected

  • Enhance protease inhibition if degradation is likely

  • Consider phosphatase treatment if phosphorylation contributes to shifts

• Perform validation experiments:

  • Use blocking peptides to confirm specificity

  • Compare wild-type and knockout samples

  • Test antibody against recombinant AT2G11200 protein

• Advanced analysis techniques:

  • Consider 2D gel electrophoresis to separate isoforms by both size and charge

  • Apply mass spectrometry to identify the exact nature of each band

Understanding the pattern of bands can provide valuable insights into AT2G11200 regulation and post-translational processing in different experimental conditions.

How can I quantitatively analyze AT2G11200 expression levels across different plant tissues or conditions?

Quantitative analysis of AT2G11200 expression requires rigorous methodology to ensure accuracy and reproducibility:

• Sample Standardization:

  • Collect tissues at consistent developmental stages

  • Standardize growth conditions, including light cycles, temperature, and nutrient availability

  • Process all samples simultaneously when possible

• Protein Extraction Optimization:

  • Use a consistent extraction protocol with a buffer suitable for nuclear proteins

  • Measure total protein concentration using Bradford or BCA assay

  • Load equal total protein amounts (typically 20-50 μg) for each sample

• Western Blot Quantification:

  • Include a loading control such as anti-Actin (plant) antibody on the same blot

  • Use fluorescent secondary antibodies for more accurate quantification

  • Verify signal linearity by running a dilution series

• Image Analysis Protocol:

  • Capture images within the linear range of detection

  • Use software like ImageJ to quantify band intensity

  • Normalize AT2G11200 signal to loading control signal

• Statistical Analysis:

  • Run at least three biological replicates

  • Apply appropriate statistical tests (e.g., ANOVA followed by Tukey's test)

  • Report both fold changes and p-values

• Complementary Approaches:

  • Validate protein expression changes with RT-qPCR for mRNA levels

  • Consider using a competitive binding assay similar to the one described for other proteins

How can I use antibodies to investigate the localization and trafficking of AT2G11200 in living plant cells?

Investigating the dynamic localization and trafficking of AT2G11200 requires sophisticated methodologies:

• Immunofluorescence Microscopy:

  • Fix tissues with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100

  • Use AT2G11200 antibody as primary and fluorophore-conjugated secondary antibody

  • Counterstain with DAPI to visualize nuclei (expected primary location)

  • Co-stain with markers for specific subcellular compartments

• Cell Fractionation Validation:

  • Perform subcellular fractionation to isolate nuclear, cytoplasmic, and other fractions

  • Analyze fractions by Western blot with AT2G11200 antibody

  • Use compartment-specific markers (histone H3 for nucleus, etc.) as controls

  • Compare experimental results with SUBAcon prediction (nuclear localization, 0.651)

• Proximity Labeling Approaches:

  • Create fusion proteins with BioID or APEX2 proximity labeling enzymes

  • Validate fusion proteins using AT2G11200 antibodies

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Live-Cell Imaging Complementation:

  • Validate fluorescent protein fusions with immunofluorescence using AT2G11200 antibodies

  • Use techniques like FRAP (Fluorescence Recovery After Photobleaching) to study protein dynamics

  • Confirm findings with antibody staining at fixed timepoints

• Environmental Response Analysis:

  • Investigate localization changes under various stresses or hormone treatments

  • Quantify nuclear/cytoplasmic ratios under different conditions

  • Correlate localization changes with function

This multi-faceted approach allows researchers to build a comprehensive understanding of AT2G11200's spatial regulation and movement within plant cells.

What strategies can I use to develop a highly specific monoclonal antibody against AT2G11200?

Developing a highly specific monoclonal antibody against AT2G11200 requires careful planning and execution:

• Antigen Design Considerations:

  • Analyze AT2G11200 sequence for unique regions that differ from homologous F-box proteins

  • Consider using:
    a) Full-length recombinant protein for maximum epitope coverage
    b) Unique peptide sequences (15-25 amino acids) for improved specificity
    c) Structural epitopes if 3D information is available

  • Avoid regions with high post-translational modification potential unless specifically targeting those forms

• Production Platforms:

  • Consider newer AI-guided antibody generation approaches like MAGE (Monoclonal Antibody GEnerator) that can design paired heavy-light chain antibody sequences

  • Traditional hybridoma technology remains effective for producing mouse monoclonal antibodies

  • Phage display libraries can offer alternatives when immunization is challenging

• Screening Strategy:

  • Perform initial ELISA screening against the immunizing antigen

  • Follow with Western blot screening against plant extracts

  • Include competition assays with free antigen to confirm specificity

  • Test against AT2G11200 knockout plant extracts as negative controls

• Epitope Mapping:

  • Determine the exact binding region using overlapping peptides

  • Validate binding to native protein in plant extracts

  • Ensure the epitope is accessible in applications of interest

• Validation in Multiple Applications:

  • Test antibody performance in Western blot, immunoprecipitation, immunohistochemistry

  • Determine optimal working dilutions for each application

  • Validate with recombinant protein and plant tissue samples

• Clone Selection and Production:

  • Select hybridoma clones based on specificity, affinity, and application performance

  • Ensure stable antibody production through multiple passages

  • Consider isotype selection based on application requirements (e.g., IgG2b works well for many plant applications)

This methodical approach increases the likelihood of generating a high-quality monoclonal antibody suitable for diverse research applications involving AT2G11200.

How should I design experiments to study AT2G11200 function using antibody-based approaches?

Designing robust experiments to investigate AT2G11200 function requires:

• Experimental Controls:

  • Positive Controls: Include tissues with known AT2G11200 expression

  • Negative Controls: Use at2g11200 knockout/knockdown lines

  • Antibody Controls: Include secondary-only controls and isotype controls

  • Treatment Controls: Include appropriate vehicle controls for any treatments

• Experimental Approaches Matrix:

• Experimental Design Principles:

  • Use appropriate statistical design (randomization, blocking, replication)

  • Include time-course analyses when studying dynamic processes

  • Consider tissue-specific and developmental stage-specific analyses

  • Use both loss-of-function and gain-of-function approaches for validation

• Data Integration Strategy:

  • Correlate protein levels with phenotypic data

  • Integrate with publicly available transcriptomic/proteomic datasets

  • Consider how AT2G11200 functions within known F-box protein networks

This structured approach facilitates meaningful discoveries about AT2G11200 function while minimizing experimental artifacts and misinterpretations.

What are the best methods to validate AT2G11200 antibody specificity in plant tissues?

Validating antibody specificity is crucial for generating reliable data. For AT2G11200 antibodies, consider:

• Genetic Validation Approaches:

  • Test antibody on at2g11200 knockout/knockdown lines (should show absent/reduced signal)

  • Test on AT2G11200 overexpression lines (should show increased signal)

  • Use CRISPR-Cas9 edited lines with epitope modifications

• Biochemical Validation Methods:

  • Perform peptide competition assays using the immunizing peptide

  • Pre-absorb antibody with recombinant AT2G11200 protein

  • Test cross-reactivity with closely related F-box proteins

• Specificity Testing Matrix:

• Application-Specific Validation:

  • For immunohistochemistry: Compare with RNA in situ hybridization patterns

  • For IP: Confirm pulled-down protein identity by mass spectrometry

  • For Western blot: Compare migration with recombinant protein standard

• Cross-Species Validation:

  • If the antibody is expected to recognize homologs in other species, test against extracts from those species

  • Compare observed patterns with sequence conservation data

These comprehensive validation approaches ensure that experimental findings using AT2G11200 antibodies are reliable and reproducible across different research contexts.

How can I combine AT2G11200 antibodies with emerging technologies to study dynamic protein regulation?

Integrating AT2G11200 antibodies with cutting-edge technologies provides powerful insights into dynamic regulation:

• Proximity-Dependent Labeling:

  • Validate BioID or TurboID fusion constructs using AT2G11200 antibodies

  • Map the dynamic interactome of AT2G11200 under different conditions

  • Identify transient interactions that may be missed by traditional co-IP

• Single-Cell Protein Analysis:

  • Apply AT2G11200 antibodies in single-cell Western blot technologies

  • Combine with single-cell transcriptomics to correlate protein and mRNA levels

  • Analyze cell-to-cell variability in AT2G11200 expression and localization

• Super-Resolution Microscopy:

  • Use fluorophore-conjugated secondary antibodies compatible with STORM/PALM

  • Achieve nanoscale resolution of AT2G11200 localization within the nucleus

  • Co-visualize with other proteins to map nuclear subdomains

• Optogenetic Approaches:

  • Validate optogenetic fusion constructs with AT2G11200 antibodies

  • Study real-time protein relocalization followed by fixation and immunostaining

  • Correlate light-induced functional changes with protein dynamics

• Quantitative Multiplexed Imaging:

  • Use AT2G11200 antibodies in CycIF (Cyclic Immunofluorescence) or CODEX

  • Profile dozens of proteins simultaneously in the same tissue section

  • Create comprehensive spatial maps of F-box protein networks

These integrated approaches push beyond traditional antibody applications to reveal dynamic aspects of AT2G11200 biology that would otherwise remain inaccessible.

What are the considerations for applying AT2G11200 antibodies in cross-species studies of F-box protein conservation?

Applying AT2G11200 antibodies across species requires careful methodological considerations:

• Epitope Conservation Analysis:

  • Perform sequence alignment of AT2G11200 homologs across species

  • Identify regions of high conservation that may contain shared epitopes

  • Consider generating antibodies to highly conserved regions for cross-species studies

• Experimental Validation Strategy:

  • Test antibodies systematically against extracts from multiple species

  • Validate bands of interest by mass spectrometry when possible

  • Use genetic approaches (when available) to confirm specificity

• Cross-Reactivity Assessment Matrix:

• Optimization for Diverse Plant Tissues:

  • Adjust extraction protocols for different plant species (cell wall composition varies)

  • Optimize antibody concentration and incubation conditions for each species

  • Consider using plant-specific detection systems like anti-actin antibodies as controls

• Functional Conservation Studies:

  • Combine immunolocalization across species with functional assays

  • Correlate protein expression patterns with conserved phenotypes

  • Integrate with evolutionary analyses of F-box gene families

This methodical approach allows researchers to leverage AT2G11200 antibodies for comparative studies that illuminate evolutionary conservation and divergence of F-box protein functions across plant species.