At2g19630 Antibody

What is At2g19630 and what does the antibody target?

At2g19630 refers to a gene in Arabidopsis thaliana that encodes an F-box and associated interaction domains-containing protein according to the Araport11 database . The antibody against At2g19630 is designed to specifically recognize and bind to this protein. F-box proteins are crucial components of the SCF (Skp1-Cullin-F-box) complex, which facilitates protein ubiquitination and subsequent degradation through the proteasome pathway. These proteins play essential roles in various cellular processes including cell cycle regulation, signal transduction, and developmental processes in plants.

The At2g19630 antibody is typically a rabbit polyclonal antibody purified by antigen affinity chromatography, designed to detect this specific F-box protein in plant samples . The target epitope is generally derived from recombinant Arabidopsis thaliana At2g19630 protein, allowing for specific detection in experimental applications.

What experimental applications is the At2g19630 antibody validated for?

The At2g19630 antibody has been validated primarily for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blotting (WB) applications . These techniques enable researchers to detect, quantify, and analyze the protein in various experimental contexts:

• Western Blotting: Allows for the detection of At2g19630 protein in complex mixtures, providing information about protein size, abundance, and potential modifications

• ELISA: Enables quantitative analysis of At2g19630 protein levels in samples

• Immunohistochemistry/Immunofluorescence: May be feasible, though specific validation for these applications should be confirmed

When designing experiments with this antibody, researchers should consider species reactivity (primarily plant species, particularly Arabidopsis thaliana) and conduct appropriate validation steps to ensure specificity in their experimental system .

How should I validate the specificity of At2g19630 antibody?

Proper antibody validation is critical for ensuring reliable experimental results. Based on enhanced validation methods in the literature , implement these approaches for At2g19630 antibody:

Orthogonal Validation:

• Compare antibody-based detection with an antibody-independent method like mass spectrometry

• Correlate protein expression with mRNA levels using qRT-PCR or RNA-seq data

Genetic Validation:

• Test the antibody in tissues from knockout/knockdown mutants where At2g19630 expression is absent or reduced

• Use CRISPR-Cas9 edited plants lacking At2g19630 as negative controls

Independent Antibody Validation:

• Use multiple antibodies targeting different epitopes of At2g19630

• Compare staining patterns between these independent antibodies

Pre-adsorption Test:

• Pre-incubate the antibody with purified antigen before application

• If the antibody is specific, pre-adsorption should eliminate or significantly reduce the signal

What controls should I include when using At2g19630 antibody?

Incorporating appropriate controls is essential for interpreting antibody-based experiments correctly:

Essential Controls for At2g19630 Antibody Experiments:

For Western blot experiments specifically, include a molecular weight marker to confirm the target protein's expected size, and consider using recombinant At2g19630 protein as a positive control when available . For immunohistochemistry, include tissues known to not express the protein as negative controls.

Research on anti-therapeutic antibodies has demonstrated that preexisting antibodies can sometimes interfere with results, so appropriate competition assays may be necessary in some experimental contexts to distinguish specific from non-specific signals .

What is the recommended protocol for using At2g19630 antibody in Western blotting?

For optimal Western blotting results with At2g19630 antibody, follow this methodological approach:

Sample Preparation:

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

• Quantify protein concentration using Bradford or BCA assay

• Prepare samples containing 20-50 μg of total protein per lane

• Denature samples by heating at 95°C for 5 minutes in Laemmli buffer

SDS-PAGE and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels (F-box proteins typically range from 40-60 kDa)

• Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight at 4°C

Immunodetection:

• Block membrane with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

• Incubate with At2g19630 antibody (start with 1:1000 dilution, optimize as needed) overnight at 4°C

• Wash 3-5 times with TBST, 5 minutes each

• Incubate with appropriate secondary antibody (e.g., anti-rabbit HRP if the primary is rabbit-derived) for 1 hour at room temperature

• Wash 3-5 times with TBST, 5 minutes each

• Develop using ECL substrate and detect signal using film or digital imager

Controls:

• Include wild-type and At2g19630 knockout/knockdown samples if available

• Consider running recombinant At2g19630 protein as a positive control

• Include a loading control (e.g., anti-actin or anti-tubulin) to normalize protein loading

This protocol may require optimization based on specific research conditions. Document any modifications to establish reproducible procedures for your experimental system.

How should I optimize the concentration of At2g19630 antibody for my specific application?

Optimizing antibody concentration is essential for achieving the best signal-to-noise ratio in your experiments:

Titration Experiment Strategy:

• Prepare a dilution series of the antibody (e.g., 1:100, 1:500, 1:1000, 1:2000, 1:5000)

• Keep all other experimental conditions constant

• Process samples in parallel

• Evaluate results based on signal strength and background levels

For Western Blotting:

• Start with manufacturer's recommended dilution (typically 1:1000)

• If signal is too strong with high background, increase dilution

• If signal is weak, decrease dilution or extend exposure time

• Consider the detection method (ECL vs. fluorescence) when optimizing

For Immunohistochemistry/Immunofluorescence:

• Begin with a moderate dilution (e.g., 1:200-1:500)

• Assess both signal intensity and background in positive and negative control tissues

• Incrementally adjust until optimal staining is achieved

Documentation Table Format:

The optimal concentration will provide maximum specific signal with minimal background. Document your optimization process thoroughly to ensure reproducibility in future experiments.

How can I use At2g19630 antibody for protein localization studies?

For protein localization studies using the At2g19630 antibody, implement these methodological approaches:

Subcellular Fractionation Approach:

• Isolate nuclear, cytoplasmic, membrane, and organelle fractions using differential centrifugation

• Analyze fractions by Western blotting using At2g19630 antibody

• Include fraction-specific markers (e.g., histone H3 for nuclear, tubulin for cytoplasmic)

• Quantify relative distribution across cellular compartments

Immunofluorescence Microscopy Protocol:

• Fix Arabidopsis tissues or protoplasts using 4% paraformaldehyde

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 3% BSA in PBS

• Incubate with At2g19630 antibody (optimized dilution)

• Use fluorophore-conjugated secondary antibody for detection

• Include DAPI for nuclear staining

• Analyze using confocal microscopy

Co-localization Analysis:

• Perform double immunolabeling with antibodies against known subcellular markers

• Calculate co-localization coefficients (Pearson's, Mander's)

• Consider using specific organelle markers (e.g., ER, Golgi, vacuole)

Research on the ATG6 protein demonstrates the importance of examining both nuclear and cytoplasmic fractions separately when studying plant regulatory proteins . For example, researchers found that ATG6 co-localized with NPR1 in the nucleus, and SA treatment promoted both cytoplasmic and nuclear accumulation of ATG6, providing important insights into protein function .

How can At2g19630 antibody be utilized in studies of protein-protein interactions?

When investigating protein-protein interactions with At2g19630 antibody, implement these methodological approaches:

Co-Immunoprecipitation (Co-IP) Protocol:

• Harvest and lyse plant tissue in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, and protease inhibitors

• Clear lysate by centrifugation (14,000 × g, 10 min, 4°C)

• Pre-clear with Protein A/G beads (1 hour, 4°C)

• Incubate cleared lysate with At2g19630 antibody overnight at 4°C

• Add Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads 4-5 times with lysis buffer

• Elute proteins by boiling in SDS sample buffer

• Analyze by SDS-PAGE and immunoblotting with antibodies against suspected interacting proteins

Proximity Ligation Assay (PLA):

• Fix plant cells or tissue sections

• Incubate with At2g19630 antibody and antibody against putative interacting protein

• Use PLA probes specific to the primary antibody species

• Perform ligation and amplification according to manufacturer's protocol

• Visualize interaction signals using fluorescence microscopy

• Quantify PLA signals per cell using appropriate software

Considerations Specific to At2g19630:

• F-box proteins typically interact with SKP1, Cullin, and substrate proteins

• Buffer conditions may need optimization to maintain these interactions

• Consider the potential impact of post-translational modifications on interactions

Research on protein interactions in plants demonstrates how careful antibody-based co-localization and co-immunoprecipitation studies can reveal important functional relationships . For example, studies of ATG6-NPR1 interactions revealed that ATG6 directly interacts with NPR1 and significantly increases nuclear accumulation of NPR1, providing insight into the regulatory mechanisms of plant immunity .

How can I use At2g19630 antibody in combination with other techniques for comprehensive protein analysis?

Integrating multiple techniques with antibody-based detection provides more robust and comprehensive protein analysis:

Mass Spectrometry Integration:

• Use immunoprecipitation with At2g19630 antibody followed by MS analysis

• Identify interacting proteins and post-translational modifications

• Validate MS findings with reciprocal co-immunoprecipitation

• Analyze ubiquitination patterns (particularly relevant for F-box proteins)

Multi-omics Approach:

• Correlate protein expression (antibody-based) with transcriptomics data

• Integrate with metabolomics to understand functional consequences

• Consider proteogenomics to identify novel protein variants

• Example experimental design:

Proximity Labeling:

• Combine with BioID or APEX2 techniques to identify proximal proteins

• Express BioID-At2g19630 fusion in plants

• Purify biotinylated proteins

• Confirm interactions using At2g19630 antibody

Functional Assays:

• Pair antibody detection with ubiquitination assays

• Correlate protein levels with phenotypic outcomes

• Use the antibody to immunodeplete At2g19630 and assess functional consequences

Based on protein microarray research, combining antibody detection with orthogonal methods significantly enhances data reliability and biological insights . As demonstrated in studies of recombinant antibody production in Arabidopsis, integrating antibody-based detection with transcriptome analysis via Tiling arrays provides a comprehensive view of protein expression and regulation .

How should I quantify and analyze data obtained using At2g19630 antibody?

Western Blot Quantification Protocol:

• Use digital imaging systems rather than film for better dynamic range

• Capture images before signal saturation occurs

• Define regions of interest (ROIs) for specific bands and background

• Subtract background from each band's intensity

• Normalize target protein bands to loading controls (e.g., actin, GAPDH)

• Calculate relative expression as: (At2g19630 signal / loading control signal)

• Compare across conditions using fold change relative to control

Immunohistochemistry Quantification:

• Use consistent exposure settings across all samples

• Quantify signal intensity across multiple regions of interest (minimum 5-10 per condition)

• Measure nuclear vs. cytoplasmic distribution for localization studies

• Apply appropriate thresholding to distinguish specific from non-specific signal

• Consider cell-by-cell analysis for heterogeneous tissues

ELISA Data Analysis:

• Generate standard curves using purified protein if available

• Ensure measurements fall within the linear range of detection

• Run samples in technical triplicates to assess variability

• Calculate concentration based on standard curve regression

According to research on antibody microarrays, proper normalization procedures that eliminate systematic bias are crucial for accurate interpretation . For antibody arrays, similar statistical methods as those used for cDNA arrays can be applied, including background subtraction, normalization, and differential expression analysis to identify significant changes in protein abundance across conditions .

What statistical methods are appropriate for analyzing results from experiments using At2g19630 antibody?

Selecting appropriate statistical methods depends on your experimental design and data characteristics:

Statistical Analysis Recommendations by Experiment Type:

Important Statistical Considerations:

• Apply multiple testing correction (Bonferroni, FDR) when performing numerous comparisons

• Verify data normality (Shapiro-Wilk test) before selecting parametric tests

• Consider non-parametric alternatives for non-normal data

• Report exact p-values and confidence intervals, not just significance level

• Include effect sizes to indicate biological relevance

How can I address contradictory results when using At2g19630 antibody across different experimental conditions?

Contradictory results are common in antibody-based research and require systematic investigation:

Systematic Troubleshooting Approach:

• Reassess Antibody Validation:

  • Re-validate antibody specificity using orthogonal methods

  • Test for lot-to-lot variability if using different antibody batches

  • Consider epitope accessibility issues in different experimental conditions

  • Verify antibody storage conditions and expiration dates

• Analyze Experimental Conditions:

  • Document all variables between experiments (buffers, incubation times, temperatures)

  • Test critical parameters individually to identify sources of variation

  • Consider post-translational modifications that might affect antibody binding

  • Evaluate sample preparation differences (lysis buffers, fixation methods)

• Evaluate Biological Variables:

  • Assess developmental stages, tissue specificity, or stress conditions

  • Consider circadian or seasonal effects on protein expression

  • Evaluate genetic background differences between sample sources

  • Account for environmental factors that may influence protein expression

• Implement Reconciliation Strategies:

  • Employ orthogonal methods to confirm findings (MS, functional assays)

  • Use alternative antibodies targeting different epitopes

  • Consider targeted genetic approaches (RNAi, CRISPR) to validate findings

  • Design experiments that directly test competing hypotheses

According to studies on antibody validation, contradictory results often stem from insufficient validation or variable experimental conditions . Enhanced validation methods emphasize using orthogonal approaches and independent antibodies to increase confidence in results. Research on antibody quality has revealed that approximately 50% of commercial antibodies fail to meet basic standards for characterization, contributing to reproducibility issues in biological research .

How can At2g19630 antibody be utilized in studies of plant stress responses?

At2g19630 encodes an F-box protein in Arabidopsis, which likely participates in protein degradation pathways that regulate stress responses. Here's how to utilize the antibody in stress response studies:

Temporal Expression Analysis Protocol:

• Expose plants to specific stressors (drought, salt, pathogen, heat)

• Harvest tissue at multiple timepoints (0, 1, 3, 6, 12, 24, 48 hours)

• Extract proteins using buffer optimized for plant tissues

• Analyze At2g19630 protein levels by Western blotting

• Quantify relative to unstressed controls

• Compare protein dynamics with mRNA expression patterns using qRT-PCR

Spatial Distribution Studies:

• Use immunohistochemistry to determine tissue-specific expression changes

• Investigate subcellular relocalization under stress conditions

• Compare root, stem, leaf, and reproductive tissues for differential responses

• Analyze protein distribution changes in response to localized vs. systemic stress

Protein Degradation Dynamics:

• Use cycloheximide chase assays to determine protein stability under stress

• Investigate ubiquitination patterns using immunoprecipitation followed by ubiquitin-specific antibodies

• Examine proteasome involvement using inhibitors like MG132

• Track protein half-life changes in response to different stress conditions

Studies on plant transcription factors like WRKY75 , which may function in the same pathways as At2g19630, provide insights into how regulatory proteins respond to various stresses. Research has identified numerous target genes of WRKY75, suggesting complex regulatory networks that may also involve F-box proteins like At2g19630 in stress response pathways .

What approaches can I use to study At2g19630 protein degradation mechanisms?

As an F-box protein, At2g19630 likely functions in the ubiquitin-proteasome system to target substrate proteins for degradation. Here are methodological approaches to study these mechanisms:

Substrate Identification Protocol:

• Generate transgenic plants expressing tagged At2g19630 (e.g., FLAG, HA, GFP)

• Perform immunoprecipitation using antibodies against the tag

• Identify co-precipitating proteins by mass spectrometry

• Validate potential substrates using directed co-IP with At2g19630 antibody

• Confirm interaction specificity using at2g19630 knockout controls

Ubiquitination Assay:

• Immunoprecipitate candidate substrate proteins

• Probe with anti-ubiquitin antibodies to detect ubiquitination

• Compare ubiquitination levels between wild-type and at2g19630 mutant plants

• Use proteasome inhibitors (MG132) to accumulate ubiquitinated intermediates

Degradation Kinetics:

• Perform cycloheximide chase assays to measure substrate half-life

• Compare protein stability in wild-type vs. at2g19630 mutant backgrounds

• Quantify protein levels over time using At2g19630 antibody

• Calculate degradation rates under various conditions

SCF Complex Analysis:

• Use At2g19630 antibody to immunoprecipitate the complete SCF complex

• Identify associated Skp1 and Cullin proteins by Western blotting

• Study complex assembly/disassembly dynamics under different conditions

• Investigate post-translational modifications that regulate F-box protein activity

Research on protein degradation mechanisms in plants has shown that F-box proteins like At2g19630 can be regulated at multiple levels, including transcription, protein stability, and subcellular localization . Studying these regulatory mechanisms requires combining antibody-based detection with genetic approaches and biochemical assays to fully understand their function in plant development and stress responses.