At4g09920 Antibody

What is At4g09920 Antibody and what target does it recognize?

At4g09920 Antibody specifically recognizes the F-box protein encoded by the At4g09920 gene located on chromosome 4 of Arabidopsis thaliana. This antibody targets the T5L19.50 F-box protein (UniProt accession number Q9T0F1), which is involved in protein-protein interactions and likely participates in ubiquitin-mediated protein degradation pathways within plant cells. The antibody is supplied in liquid form with a buffer composition of 0.03% ProClin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage and handling.

What are the validated experimental applications for At4g09920 Antibody?

While specific validation data is not directly provided in the available information, based on standard practices for similar plant protein antibodies, At4g09920 Antibody is likely suitable for applications including Western blotting, immunoprecipitation, immunofluorescence, and potentially ELISA. For precise experimental validation information, researchers should request application-specific data from the manufacturer or conduct preliminary validation experiments for their specific experimental conditions. The antibody's high specificity for the target protein makes it valuable for examining protein expression, localization, and interaction studies in plant systems.

What are the optimal storage and handling conditions for At4g09920 Antibody?

For maximum stability and performance, At4g09920 Antibody should be stored at -20°C for long-term preservation. When in use, the antibody can be kept at 4°C for short periods (1-2 weeks). The product is shipped with ice packs to maintain cold chain integrity. The 50% glycerol in the buffer formulation helps prevent freeze-thaw damage, but repeated freeze-thaw cycles should still be avoided. It is advisable to prepare small working aliquots upon first thawing to minimize potential degradation. The presence of 0.03% ProClin 300 in the buffer helps maintain antibody integrity by preventing microbial contamination during handling and storage.

What protocols are recommended for Western blot applications with At4g09920 Antibody?

For Western blot applications using At4g09920 Antibody, researchers should follow this optimized protocol:

• Sample Preparation:

  • Extract total protein from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

  • Quantify protein concentration using Bradford or BCA assay

  • Prepare samples containing 20-50 μg protein per lane

• Gel Electrophoresis and Transfer:

  • Separate proteins using 10-12% SDS-PAGE

  • Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Immunoblotting:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute At4g09920 Antibody 1:500-1:2000 in blocking solution

  • Incubate membrane with diluted antibody overnight at 4°C with gentle agitation

  • Wash 3× with TBST for 10 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10,000)

  • Wash 3× with TBST

  • Detect signal using enhanced chemiluminescence

• Controls:

  • Include wild-type and At4g09920 knockout/knockdown samples if available

  • Consider using recombinant At4g09920 protein as a positive control

This protocol may require optimization based on specific experimental conditions and sample types.

How should researchers design immunoprecipitation experiments using At4g09920 Antibody?

For successful immunoprecipitation experiments with At4g09920 Antibody:

• Lysate Preparation:

  • Harvest and grind plant tissue in liquid nitrogen

  • Extract proteins in IP buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors)

  • Clear lysate by centrifugation (14,000 × g, 15 minutes, 4°C)

• Pre-clearing:

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

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of At4g09920 Antibody to 500 μl of pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 30 μl of Protein A/G beads

  • Incubate for 2-3 hours at 4°C

  • Collect beads by centrifugation

  • Wash 4× with IP buffer

  • Elute proteins by boiling in SDS sample buffer

• Analysis:

  • Analyze precipitated proteins by Western blotting or mass spectrometry

  • Use IgG of the same species as negative control

  • Include input sample (5-10% of lysate used for IP)

This approach enables the investigation of protein-protein interactions involving the At4g09920 F-box protein, particularly its role in ubiquitin-mediated protein degradation pathways.

What are the recommended approaches for immunolocalization studies with At4g09920 Antibody?

For effective immunolocalization of At4g09920 protein in plant tissues:

• Sample Preparation:

  • Fix plant tissues in 4% paraformaldehyde in PBS for 2-4 hours

  • Wash in PBS (3×, 10 minutes each)

  • For sectioning: embed in paraffin or prepare cryosections (10-20 μm thickness)

  • For whole-mount: permeabilize with 0.1-0.5% Triton X-100 in PBS

• Antigen Retrieval (if needed):

  • Heat sections in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Cool to room temperature

• Immunostaining:

  • Block with 3% BSA, 0.1% Triton X-100 in PBS for 1 hour

  • Incubate with At4g09920 Antibody (1:100-1:500 dilution) overnight at 4°C

  • Wash with PBS (3×, 10 minutes each)

  • Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature

  • Wash with PBS (3×, 10 minutes each)

  • Counterstain nuclei with DAPI (1 μg/ml) for 10 minutes

  • Mount in anti-fade mounting medium

• Controls and Validation:

  • Include secondary antibody-only control

  • Use tissue from At4g09920 knockout plants as negative control

  • Consider co-localization with known cell compartment markers

This protocol helps researchers visualize the subcellular localization of At4g09920 protein, providing insights into its functional roles within plant cells.

How can researchers troubleshoot weak or absent signal when using At4g09920 Antibody?

When facing weak or absent signals with At4g09920 Antibody, consider these methodical troubleshooting steps:

For tissue-specific experiments, consider that At4g09920 expression may vary across developmental stages and in response to environmental stimuli. Using appropriate positive controls and normalizing to loading controls are essential for accurate result interpretation.

What strategies can optimize specificity when working with At4g09920 Antibody in complex plant extracts?

To enhance specificity when detecting At4g09920 protein in complex plant extracts:

• Sample Preparation Optimization:

  • Use plant-specific extraction buffers containing polyvinylpolypyrrolidone (PVPP) to remove phenolic compounds

  • Include 5-10 mM DTT to maintain protein reduction state

  • Add specific protease inhibitors targeting plant proteases

  • Consider tissue-specific extraction protocols based on target abundance

• Antibody Specificity Enhancement:

  • Pre-absorb antibody with extracts from At4g09920 knockout plants

  • Perform competition assays with recombinant At4g09920 protein

  • Use affinity purification against the specific epitope

• Detection System Modifications:

  • Employ enhanced chemiluminescence plus (ECL+) for increased sensitivity

  • Consider using biotin-streptavidin amplification systems

  • Use highly cross-adsorbed secondary antibodies to minimize non-specific binding

• Validation Approaches:

  • Compare results with RNA expression data (qRT-PCR or RNA-seq)

  • Use genetic knockdown/knockout lines as negative controls

  • Confirm results with a second antibody recognizing a different epitope if available

Implementation of these strategies will significantly improve detection specificity of the target F-box protein in various experimental contexts.

How does post-translational modification of At4g09920 affect antibody recognition?

Post-translational modifications (PTMs) of the At4g09920 F-box protein can significantly impact antibody recognition and experimental outcomes. F-box proteins are frequently regulated by PTMs including:

• Phosphorylation:

  • Phosphorylation sites on At4g09920 may alter epitope accessibility

  • Treatment with phosphatase inhibitors during extraction is recommended to preserve phosphorylation state

  • For phosphorylation-specific studies, consider phospho-state specific antibodies if available

• Ubiquitination:

  • As an F-box protein involved in ubiquitin-mediated processes, At4g09920 may itself be ubiquitinated

  • Include deubiquitinating enzyme inhibitors (e.g., PR-619) in extraction buffers

  • For ubiquitination studies, consider immunoprecipitation followed by ubiquitin-specific Western blotting

• Other Modifications:

  • SUMOylation, neddylation, or other modifications may occur

  • These modifications can add 10-20 kDa to the apparent molecular weight

Researchers should be aware that the epitope recognized by At4g09920 Antibody may be masked by certain PTMs, potentially leading to false-negative results in tissues where the protein is heavily modified. Comparative analysis of different extraction conditions (with and without modification-preserving inhibitors) can provide insights into the regulation of this F-box protein through post-translational mechanisms.

What approaches are recommended for studying At4g09920 protein interactions with SCF complex components?

F-box proteins like At4g09920 typically function as part of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes. To study these interactions:

• Co-immunoprecipitation Strategy:

  • Use At4g09920 Antibody for immunoprecipitation followed by Western blotting for known SCF components (ASK1/SKP1, CUL1, RBX1)

  • Reverse co-IP using antibodies against SCF components to confirm interaction

  • Include appropriate negative controls (IgG and unrelated protein antibodies)

• Proximity Ligation Assay (PLA):

  • Perform in situ detection of protein-protein interactions

  • Co-incubate samples with At4g09920 Antibody and antibodies against SCF components

  • Use species-specific PLA probes and visualize interaction signals by fluorescence microscopy

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of At4g09920 and potential interacting partners

  • Transiently express in plant protoplasts or through stable transformation

  • Validate results with point mutations in interaction domains

• Mass Spectrometry-Based Approaches:

  • Perform immunoprecipitation with At4g09920 Antibody

  • Analyze precipitated proteins by LC-MS/MS

  • Confirm novel interactions by reciprocal co-IP or PLA

These approaches provide complementary evidence for protein interactions, offering insights into the functional role of At4g09920 in protein degradation pathways and plant cellular processes.

How can researchers quantitatively analyze At4g09920 protein expression across different developmental stages or stress conditions?

For quantitative analysis of At4g09920 protein expression under various conditions:

• Quantitative Western Blotting:

  • Use infrared fluorescence-based detection systems (e.g., LI-COR Odyssey)

  • Include recombinant At4g09920 protein standards at known concentrations

  • Normalize to multiple housekeeping proteins (e.g., actin, tubulin, GAPDH)

  • Analyze using appropriate software (ImageJ, Image Studio)

• ELISA-Based Quantification:

  • Develop sandwich ELISA using At4g09920 Antibody

  • Generate standard curves with recombinant protein

  • Optimize extraction conditions to maximize protein recovery

  • Use biological and technical replicates for statistical validity

• Proteomics Approach:

  • Implement Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM)

  • Design specific peptide targets unique to At4g09920

  • Use isotopically labeled synthetic peptides as internal standards

  • Analyze using mass spectrometry for absolute quantification

• Data Analysis Framework:

  • Employ statistical methods appropriate for time-series or treatment comparisons

  • Use ANOVA with post-hoc tests for multi-condition experiments

  • Create expression profiles correlating protein levels with phenotypic changes

  • Integrate with transcriptomic data to identify post-transcriptional regulation

These quantitative approaches enable precise measurement of At4g09920 protein expression changes, facilitating correlation with specific developmental transitions or stress responses in plant systems.

What are the emerging applications of At4g09920 Antibody in plant stress response research?

At4g09920 Antibody offers valuable research applications in studying plant stress responses, particularly given the regulatory roles of F-box proteins in protein turnover during stress adaptation:

• Abiotic Stress Studies:

  • Monitor At4g09920 protein stability and abundance during drought, salinity, or temperature stress

  • Investigate protein relocalization under stress conditions using immunofluorescence

  • Examine post-translational modifications in response to stress signals

  • Identify stress-specific interaction partners through differential immunoprecipitation

• Plant Immunity Research:

  • Analyze At4g09920 involvement in pathogen-triggered protein degradation pathways

  • Study temporal dynamics of At4g09920 expression during immune responses

  • Investigate potential roles in hormone signaling pathways related to defense

• Developmental Plasticity:

  • Explore At4g09920 function in development-stress interaction responses

  • Study protein abundance in different tissues during stress-induced developmental reprogramming

  • Investigate epigenetic regulation of At4g09920 during stress memory formation

• Targeted Proteomics Applications:

  • Develop At4g09920-centered protein degradation networks

  • Identify substrates whose stability is regulated by At4g09920-containing SCF complexes

  • Map changes in the At4g09920 interactome under various stress conditions

These applications highlight the value of At4g09920 Antibody in deciphering stress signaling networks and protein degradation mechanisms that underlie plant adaptation to environmental challenges.

What considerations are important when designing CRISPR-Cas9 gene editing experiments to validate At4g09920 Antibody specificity?

CRISPR-Cas9 gene editing provides powerful validation tools for antibody specificity. When designing such experiments for At4g09920:

• Guide RNA Design Strategy:

  • Target early exons to ensure complete protein disruption

  • Design multiple gRNAs (3-4) targeting different regions of At4g09920

  • Avoid off-target effects by comprehensive bioinformatic screening

  • Consider targeting the epitope region specifically recognized by the antibody

• Validation Protocol Design:

  • Generate homozygous knockout lines through selection and screening

  • Confirm gene editing by sequencing the target region

  • Perform Western blotting with At4g09920 Antibody comparing wild-type and knockout lines

  • Include heterozygous plants to assess dose-dependence of antibody signal

• Controls and Considerations:

  • Generate epitope-modified lines where possible (in-frame mutations)

  • Consider potential redundancy with closely related F-box proteins

  • Assess phenotypic effects of knockout to ensure viable tissues for testing

  • Include CRISPR control lines (Cas9 expression without gRNA)

• Complementation Testing:

  • Reintroduce wild-type At4g09920 to knockout lines

  • Confirm restoration of antibody detection

  • Consider introducing tagged versions for independent verification

This systematic approach provides definitive validation of antibody specificity while generating valuable knockout resources for functional studies of At4g09920.

How can researchers integrate At4g09920 protein expression data with transcriptomic and phenotypic analyses?

Integrative approaches combining At4g09920 protein data with other molecular and phenotypic datasets:

• Multi-Omics Data Integration:

  • Correlate protein levels (detected with At4g09920 Antibody) with corresponding mRNA levels

  • Calculate protein/mRNA ratios to identify post-transcriptional regulation

  • Incorporate metabolomic data to link protein function with metabolic outcomes

  • Develop network models incorporating protein, transcript, and metabolite data

• Phenotypic Correlation Analysis:

  • Document morphological, physiological, and developmental phenotypes

  • Establish statistical correlations between At4g09920 protein levels and phenotypic traits

  • Implement machine learning approaches for pattern recognition across datasets

  • Create predictive models of plant responses based on protein expression patterns

• Data Visualization and Analysis Platforms:

  • Utilize dimensional reduction techniques (PCA, t-SNE) for data visualization

  • Implement hierarchical clustering to identify co-regulated genes and proteins

  • Develop online resources integrating At4g09920 data across experimental conditions

  • Apply pathway enrichment analysis to contextualize At4g09920 function

• Temporal and Spatial Resolution:

  • Capture dynamic changes in At4g09920 protein expression over time and space

  • Correlate with developmental transitions or stress response phases

  • Implement time-series analysis methods for identifying regulatory relationships

  • Create mathematical models describing At4g09920 regulation and function

This integrated approach provides a comprehensive understanding of At4g09920 biology beyond what can be achieved through any single analytical approach.

What are the detailed specifications of commercially available At4g09920 Antibody?

The commercially available At4g09920 Antibody has the following specifications:

The antibody is produced as a made-to-order reagent with a lead time of 14-16 weeks, suggesting it undergoes rigorous quality control and validation procedures before shipping. Researchers planning experiments should account for this production timeframe in their experimental schedule.

What detection systems are most compatible with At4g09920 Antibody for various applications?

Optimal detection systems for At4g09920 Antibody across applications:

• Western Blotting Detection Systems:

  • Enhanced chemiluminescence (ECL) for standard sensitivity

  • ECL Plus or SuperSignal West Femto for enhanced sensitivity

  • Fluorescent secondary antibodies (IRDye 680/800) for quantitative analysis

  • Colorimetric detection (DAB or TMB) for low-cost alternatives

• Immunofluorescence Detection:

  • Alexa Fluor 488/555/647-conjugated secondary antibodies

  • Tyramide signal amplification for low-abundance targets

  • Quantum dots for enhanced photostability in long-term imaging

  • Appropriate filter sets for minimizing autofluorescence from plant tissues

• Immunohistochemistry Options:

  • HRP-polymer detection systems

  • Biotin-streptavidin amplification

  • Alkaline phosphatase systems for tissues with endogenous peroxidase activity

• Flow Cytometry Applications:

  • PE or APC-conjugated secondary antibodies

  • Multi-color compatibility considerations for co-labeling experiments

Selection of the optimal detection system should be based on the abundance of At4g09920 protein in the experimental system, the required sensitivity, and the specific technical limitations of the plant tissues being analyzed.