At2g24615 Antibody

What is the At2g24615 gene and what protein does it encode?

At2g24615 is a gene in Arabidopsis thaliana (Mouse-ear cress). The antibody (CSB-PA652771XA01DOA) specifically recognizes the protein encoded by this gene (UniProt ID: Q2V462). The protein is involved in cellular processes in Arabidopsis, and targeted antibodies enable researchers to detect, quantify, and localize this protein in various experimental contexts. Researchers studying this gene should consult current genomic databases for the most recent annotations and functional predictions, as our understanding of the Arabidopsis genome continues to evolve with new research findings .

What are the primary applications for At2g24615 Antibody in plant research?

At2g24615 Antibody can be utilized in multiple experimental applications:

• Western blotting for protein detection and quantification

• Immunofluorescence microscopy for cellular localization studies

• Immunoprecipitation for protein-protein interaction studies

• Chromatin immunoprecipitation (ChIP) for protein-DNA interaction studies

These applications are fundamental for investigating protein expression patterns, subcellular localization, and functional interactions in Arabidopsis research .

What is the recommended storage and handling protocol for At2g24615 Antibody?

For optimal performance and longevity of the At2g24615 Antibody:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by aliquoting upon receipt

• For short-term storage (1-2 weeks), keep at 4°C

• Avoid exposure to direct light

• Follow manufacturer-recommended dilution ratios for specific applications

• Always include positive and negative controls in experiments

Proper storage and handling ensure antibody activity and experimental reproducibility, which are essential for generating reliable scientific data.

How can I optimize the At2g24615 Antibody for use in different Arabidopsis tissue types?

Optimization for different Arabidopsis tissues requires systematic assessment of multiple parameters:

Begin with these parameters and perform validation experiments on each tissue type. Adjust protocols based on signal-to-noise ratio and specificity of staining patterns. Tissue-specific optimization is crucial as protein expression levels, accessibility, and cross-reactivity can vary significantly between different plant tissues .

What approaches can I use to validate the specificity of At2g24615 Antibody?

To confirm antibody specificity through a multi-faceted validation approach:

• Genetic controls: Test antibody reactivity in wild-type versus knockout/knockdown lines for At2g24615

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide to block specific binding

• Mass spectrometry analysis: Perform MS on immunoprecipitated material to confirm target identity

• Cross-reactivity testing: Test against related proteins in the same family

• Multiple antibody comparison: Use independent antibodies targeting different epitopes of the same protein

Implementing at least three of these approaches provides robust validation of antibody specificity. Researchers should document validation methods thoroughly in publications to enhance reproducibility .

How can I determine if the At2g24615 protein expression changes during developmental stages?

To study developmental expression patterns:

• Collect tissue samples from multiple developmental stages (seeds, seedlings, juvenile plants, mature plants, senescent plants)

• Prepare protein extracts using a consistent protocol for all samples

• Perform western blot analysis with At2g24615 Antibody using equal protein loading (validated by housekeeping controls)

• Quantify relative expression levels using densitometry software

• Complement protein data with RT-qPCR for transcript levels

• Consider immunofluorescence microscopy to track changes in subcellular localization

• Compare expression patterns with published RNA-seq or proteomics datasets

This multi-method approach provides comprehensive insight into temporal regulation of the target protein during development and can reveal stage-specific functions or regulatory mechanisms .

What are the optimal conditions for using At2g24615 Antibody in western blot analysis?

For western blot optimization with At2g24615 Antibody:

• Sample preparation: Extract total proteins using a buffer containing protease inhibitors

• Gel electrophoresis: Use 4-15% polyacrylamide gradient gels for optimal separation

• Protein transfer: Transfer to nitrocellulose membrane at 100V for 1 hour

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

• Primary antibody: Dilute At2g24615 Antibody 1:500 in blocking buffer, incubate overnight at 4°C

• Washing: Wash membrane three times for 5 minutes each with TBST

• Secondary antibody: Incubate with HRP-conjugated anti-mouse IgG (1:5000) for 1 hour

• Detection: Use ECL substrate and appropriate imaging system

Always include positive and negative controls, and consider running a gradient of protein concentrations to determine the linear detection range for quantitative analyses .

How do I optimize immunofluorescence protocols for At2g24615 Antibody in plant tissues?

For immunofluorescence microscopy using At2g24615 Antibody:

• Tissue fixation: Fix tissues in 4% paraformaldehyde for 12-24 hours

• Embedding: Embed in paraffin for sectioning or prepare whole-mount samples

• Sectioning: Cut 5-10 μm sections for optimal antibody penetration

• Antigen retrieval: Perform heat-induced epitope retrieval if necessary

• Blocking: Block with goat serum at 37°C for 30 minutes

• Primary antibody: Incubate with At2g24615 Antibody (1:500 dilution) overnight at 4°C

• Washing: Wash three times with PBS for 10 minutes each

• Secondary antibody: Incubate with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000) for 1 hour

• Nuclear counterstain: Stain with 1.5 mg/mL DAPI in antifade mounting medium

• Imaging: Use confocal or fluorescence microscopy with appropriate filter sets

For troubleshooting, test multiple fixation protocols and antibody concentrations to determine optimal signal-to-noise ratio. Include appropriate controls to distinguish between specific signal and autofluorescence, which is common in plant tissues .

What is the recommended protocol for immunoprecipitation with At2g24615 Antibody?

For successful immunoprecipitation with At2g24615 Antibody:

• Protein extraction: Extract proteins in non-denaturing lysis buffer with protease inhibitors

• Pre-clearing: Pre-clear lysate with protein A/G beads to reduce non-specific binding

• Antibody binding: Add At2g24615 Antibody to cleared lysate (2-5 μg antibody per 500 μg protein)

• Incubation: Incubate antibody-lysate mixture for 2 hours at 4°C

• Bead addition: Add protein A-conjugated beads and incubate for another hour

• Washing: Collect beads by centrifugation at 2000g and wash 3-5 times with TBST

• Elution: Elute bound proteins by boiling in SDS loading buffer for 10 minutes

• Analysis: Analyze by SDS-PAGE followed by western blotting or mass spectrometry

This protocol can identify interaction partners of the At2g24615 protein and confirm antibody specificity when combined with mass spectrometry analysis of immunoprecipitated material .

How can I address non-specific background when using At2g24615 Antibody?

Non-specific background can be systematically reduced through these approaches:

• Optimize blocking: Test different blocking agents (BSA, non-fat milk, normal serum) and concentrations

• Antibody titration: Perform a dilution series to determine optimal antibody concentration

• Increase washing stringency: Use higher salt concentration or add 0.1% Tween-20 to wash buffers

• Pre-adsorption: Pre-adsorb antibody with total protein from a species different from the target

• Optimize incubation conditions: Reduce incubation temperature or time

• Secondary antibody optimization: Ensure secondary antibody is compatible and properly diluted

Systematically test each parameter while keeping others constant to identify the specific factor contributing to background. Document all optimization steps for future reference and reproducibility .

How do I interpret band patterns when At2g24615 Antibody shows multiple bands?

Multiple bands may have biological or technical origins:

• Expected molecular weight: Confirm the predicted molecular weight of native At2g24615 protein

• Post-translational modifications: Consider whether modifications (phosphorylation, glycosylation) might cause molecular weight shifts

• Proteolytic processing: Determine if the protein undergoes cleavage producing multiple fragments

• Isoforms: Check databases for alternative splice variants or isoforms

• Non-specific binding: Validate specificity through peptide competition assays or knockout controls

• Protein complexes: If using non-denaturing conditions, complexes may remain intact

To confirm band identity, consider:

• Peptide competition assays

• Immunoprecipitation followed by mass spectrometry

• Comparison with recombinant protein standards

• Testing in genetic knockout/knockdown lines

A systematic approach to band interpretation prevents misattribution of signals and ensures accurate data interpretation .

How can I quantify protein expression levels using At2g24615 Antibody?

For accurate protein quantification:

• Sample preparation standardization: Use consistent extraction methods across all samples

• Equal protein loading: Validate with total protein stains (Ponceau S) and housekeeping controls

• Linear detection range: Determine the linear range of detection for the antibody

• Technical replicates: Perform at least three technical replicates per sample

• Densitometry: Use software like ImageJ to quantify band intensity

• Normalization: Normalize to appropriate reference proteins (e.g., actin, tubulin)

• Statistical analysis: Apply appropriate statistical tests to determine significance

When analyzing developmental or treatment time series, maintain consistent exposure times during imaging and process all samples simultaneously to minimize batch effects .

How can I combine At2g24615 Antibody with mass spectrometry for proteomic studies?

Integrating antibody-based approaches with mass spectrometry:

• Immunoprecipitation-mass spectrometry (IP-MS): Use the antibody to enrich the target protein and its interactors, then analyze by MS

• Sample preparation: After IP, run samples on SDS-PAGE followed by silver staining

• Band excision: Excise relevant bands for gel digestion with trypsin

• MS analysis: Analyze extracted peptides using LC-MS/MS

• Data analysis: Use appropriate software (e.g., Mascot, MaxQuant) to identify proteins

• Verification: Confirm interactions through reciprocal IPs or other methods

This approach has successfully identified protein interactors in Arabidopsis research, including identification of FtsH protease 11, glycine cleavage T-protein, and casein lytic proteinase B4 in previous studies .

How can I use At2g24615 Antibody in conjunction with genetic approaches?

Combining antibody-based and genetic approaches provides powerful insights:

• Expression correlation: Compare protein levels (antibody detection) with transcript levels (RT-qPCR)

• Mutant analysis: Examine protein expression in knockout, knockdown, or overexpression lines

• Tissue-specific expression: Compare antibody staining patterns with promoter-reporter constructs

• Protein-protein interactions: Validate interactions identified through yeast two-hybrid or BiFC using co-immunoprecipitation

• ChIP-seq integration: Combine ChIP using the antibody with RNA-seq data to correlate binding with expression

This multi-omics approach provides a comprehensive understanding of protein function within its biological context and is particularly valuable for studying proteins of unknown function .

What approaches can I use to study At2g24615 protein in different Arabidopsis ecotypes and mutant backgrounds?

For comparative studies across genetic backgrounds:

• Standardized protocols: Use identical extraction and detection methods across all samples

• Positive controls: Include wild-type Col-0 as reference in all experiments

• Protein normalization: Validate equal loading with multiple housekeeping controls

• Expression quantification: Use densitometry for western blots or fluorescence intensity measurements for immunolocalization

• Subcellular localization: Compare localization patterns across backgrounds

• Functional complementation: Test whether expression of At2g24615 in mutant backgrounds rescues phenotypes

This approach allows researchers to determine whether genetic background influences protein expression level, localization, or post-translational modifications, providing insight into genotype-dependent regulation mechanisms .

What are emerging applications for At2g24615 Antibody in plant science research?

Emerging applications include:

• Single-cell proteomics: Using the antibody for flow cytometry to quantify protein levels in isolated plant cells or protoplasts

• Super-resolution microscopy: Applying new imaging techniques like STORM or PALM for nanoscale localization

• In vivo dynamics: Correlating antibody-based detection with live-cell imaging using fluorescent fusion proteins

• Stress response studies: Examining protein expression and localization changes under various abiotic and biotic stresses

• Cross-species comparison: Using the antibody to detect homologous proteins in related plant species

These approaches expand the utility of At2g24615 Antibody beyond traditional applications and enable new insights into protein function in complex biological contexts.

How should I report At2g24615 Antibody usage in scientific publications?

For reproducible research, publications should include:

• Complete antibody identification: CSB-PA652771XA01DOA, manufacturer (CUSABIO), lot number

• Validation methods: Detailed description of specificity validation experiments

• Experimental conditions: Complete protocols including dilutions, incubation times/temperatures

• Controls: Description of positive and negative controls

• Image acquisition: Detailed parameters for microscopy or western blot imaging

• Quantification methods: Software and parameters used for any quantitative analyses

• Raw data availability: Consider depositing original, unprocessed images in data repositories

Comprehensive reporting enhances reproducibility and allows other researchers to build upon your findings effectively .