At1g59790 Antibody

What is At1g59790 antibody and what are its key specifications?

At1g59790 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana At1g59790 protein. It targets a specific protein identified by UniProt accession number Q9XIE9. The antibody is supplied in liquid form with 50% glycerol buffer containing 0.03% Proclin 300 preservative and 0.01M PBS (pH 7.4). It has been validated for ELISA and Western blot applications and is specifically reactive to Arabidopsis thaliana. This research-grade antibody is purified using antigen affinity methods to ensure specificity .

What are the recommended storage conditions for At1g59790 antibody?

For optimal stability and performance, store At1g59790 antibody at -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency. For short-term storage (less than one month), the antibody can be kept at 4°C. When handling, briefly centrifuge before opening the vial and maintain sterile conditions. Proper storage is essential for maintaining antibody specificity and sensitivity over time .

What applications has At1g59790 antibody been validated for?

At1g59790 antibody has been primarily validated for ELISA and Western blot applications. In Western blot analysis, it can detect the target protein for identification and semi-quantitative analysis. For ELISA applications, this antibody demonstrates specific binding to its target antigen. While not specifically validated for immunocytochemistry, research with other Arabidopsis antibodies suggests that affinity-purified antibodies often perform well in immunolocalization studies after proper optimization .

What is the recommended protocol for using At1g59790 antibody in Western blot applications?

For successful Western blot detection with At1g59790 antibody, follow this optimized protocol:

• Sample preparation:

  • Extract proteins from Arabidopsis tissues using appropriate buffer (typically containing detergents, protease inhibitors, and reducing agents)

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Separate 20-50 μg protein by SDS-PAGE

  • Transfer proteins to PVDF or nitrocellulose membrane

  • Verify transfer with Ponceau S staining

• Immunoblotting:

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

  • Incubate with At1g59790 antibody (starting dilution 1:1000) overnight at 4°C

  • Wash 3 times with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit, 1:5000) for 1 hour

  • Wash 3 times with TBST, 10 minutes each

  • Develop using enhanced chemiluminescence (ECL) substrate

Research shows that affinity purification significantly improves antibody performance, with properly purified Arabidopsis antibodies showing up to 55% successful detection rates compared to much lower rates with crude antisera .

How should researchers validate the specificity of At1g59790 antibody in their experimental system?

Thorough validation of At1g59790 antibody specificity is essential using multiple complementary approaches:

• Genetic validation:

  • Compare signal between wild-type and At1g59790 knockout/knockdown mutants

  • Test in overexpression lines to confirm increased signal intensity

  • Include appropriate genetic controls in all experiments

• Biochemical validation:

  • Perform peptide competition assay by pre-incubating antibody with excess immunizing peptide

  • Verify that detected protein band matches predicted molecular weight

  • Test cross-reactivity with purified recombinant protein

• Technical controls:

  • Include no-primary antibody control

  • Test pre-immune serum when available

  • Use positive control tissues with known expression

Research on Arabidopsis antibodies demonstrates that validation against respective mutant backgrounds provides the strongest confirmation of specificity. Several antibodies in the Nottingham Arabidopsis Stock Centre collection have been validated using this approach .

What troubleshooting approaches are recommended when At1g59790 antibody shows weak or no signal?

When encountering detection issues with At1g59790 antibody, implement these methodical troubleshooting steps:

• Antibody conditions optimization:

  • Test different antibody concentrations (1:500 to 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (BSA vs. milk)

• Sample preparation refinement:

  • Ensure fresh tissue extraction with complete protease inhibitors

  • Modify extraction buffer composition (test different detergents)

  • Increase protein loading (up to 50-75 μg)

• Detection system enhancement:

  • Use high-sensitivity ECL substrate

  • Increase exposure time incrementally

  • Consider signal amplification methods

• Technical parameters:

  • Optimize transfer conditions (time/voltage)

  • Test different membrane types (PVDF often provides better sensitivity)

  • Ensure appropriate secondary antibody dilution

Research with Arabidopsis antibodies shows that affinity purification significantly improves detection success, with purified antibodies showing dramatically better results compared to crude antisera .

How can At1g59790 antibody be optimized for immunolocalization studies in plant tissues?

Optimizing At1g59790 antibody for immunolocalization requires methodical protocol development:

• Tissue preparation:

  • Test multiple fixatives (4% paraformaldehyde, glutaraldehyde combinations)

  • Compare embedding methods (paraffin vs. resin vs. cryosectioning)

  • Evaluate antigen retrieval methods if needed

• Antibody parameters:

  • Determine optimal antibody concentration (start at 1:100-1:500)

  • Test extended incubation periods (overnight at 4°C)

  • Compare different blocking solutions (BSA, normal serum, milk)

• Detection system:

  • Select appropriate fluorophore-conjugated secondary antibodies

  • Consider signal amplification systems for weak signals

  • Test counterstains for structural context

• Controls:

  • Include wild-type vs. mutant tissue comparisons

  • Perform primary antibody omission controls

  • Include pre-immune serum control

Research on Arabidopsis antibodies indicates that of properly affinity-purified antibodies, approximately 22 out of 38 (58%) achieve immunocytochemistry-grade performance, demonstrating the importance of antibody purification and protocol optimization .

What are the considerations for using At1g59790 antibody in co-immunoprecipitation studies?

Implementing At1g59790 antibody for co-immunoprecipitation (Co-IP) studies requires specific methodological considerations:

• Extraction optimization:

  • Use mild, non-denaturing lysis buffers to preserve protein-protein interactions

  • Test different detergent types and concentrations (0.1-1% NP-40, Triton X-100)

  • Include stabilizing agents (10% glycerol, protease inhibitors)

  • Consider reversible crosslinking for transient interactions

• Immunoprecipitation strategy:

  • Compare direct antibody coupling to beads vs. protein A/G capture

  • Determine optimal antibody:lysate ratio through titration

  • Pre-clear lysates with beads alone to reduce background

  • Include appropriate negative controls (IgG, pre-immune serum)

• Interaction analysis:

  • Confirm successful target immunoprecipitation first

  • Test known or suspected interacting partners by immunoblotting

  • Consider mass spectrometry for unbiased partner identification

  • Validate key interactions with reciprocal Co-IP

While not specifically mentioned for At1g59790, research with other Arabidopsis antibodies demonstrates their value for understanding protein-protein interactions and regulatory networks in plant systems .

How can researchers integrate At1g59790 antibody data with other -omics approaches?

Integrating antibody-derived data with other -omics techniques provides comprehensive insights into At1g59790 function:

• Multi-level experimental design:

  • Collect matched samples for parallel analysis across platforms

  • Include appropriate time points to capture dynamic processes

  • Maintain consistent experimental conditions across analyses

  • Design statistical power for meaningful comparisons

• Integration approaches:

  • Compare protein levels (Western blot) with transcript abundance (RNA-seq)

  • Correlate protein localization with metabolite distribution

  • Analyze protein interactions in context of transcriptional networks

  • Link protein expression patterns to phenotypic outcomes

• Computational integration:

  • Apply correlation analyses across datasets

  • Implement network biology approaches

  • Use machine learning for pattern recognition

  • Develop pathway models incorporating multiple data types

The creation of comprehensive antibody resources for Arabidopsis, as described in the literature, specifically aims to facilitate systems biology approaches by enabling better understanding of protein localization and function within cell and tissue dynamics .

What protein extraction methods are most effective when working with At1g59790 antibody?

Optimizing protein extraction is critical for successful detection with At1g59790 antibody:

• Basic extraction buffer formulation:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100 or 0.1% SDS

  • 1 mM EDTA

  • 1 mM DTT

  • Complete protease inhibitor cocktail

• Extraction procedure:

  • Grind tissue thoroughly in liquid nitrogen

  • Add extraction buffer (3-5 ml per gram tissue)

  • Homogenize completely

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

  • Collect supernatant for analysis

• Method optimization:

  • Test buffer variations for target enrichment

  • Consider subcellular fractionation if appropriate

  • Evaluate protein yield and quality by SDS-PAGE

  • Assess extract stability under different storage conditions

Research with Arabidopsis antibodies demonstrates that extraction methods significantly impact detection success, with proper sample preparation being essential for reliable results .

What experimental design considerations are necessary when studying At1g59790 protein dynamics under different conditions?

Designing experiments to study At1g59790 dynamics across conditions requires methodical planning:

This approach ensures robust detection of condition-dependent changes in At1g59790 protein levels or modifications. Research on Arabidopsis antibodies emphasizes their value in understanding protein dynamics across different conditions and developmental stages .

How should researchers approach cross-species applications of At1g59790 antibody?

When considering At1g59790 antibody use beyond Arabidopsis thaliana, implement this systematic approach:

• Sequence analysis:

  • Perform sequence alignment of At1g59790 with homologs in target species

  • Focus specifically on the immunogenic region used for antibody production

  • Calculate percent identity and similarity across species

  • Consider minimum threshold of 70% amino acid identity for potential cross-reactivity

• Preliminary testing:

  • Begin with Western blot validation in the new species

  • Include Arabidopsis samples as positive control

  • Test multiple antibody concentrations

  • Compare expected vs. observed molecular weights

• Application optimization:

  • Adjust protocols based on preliminary results

  • Modify extraction buffers for species-specific requirements

  • Consider longer incubation times for cross-species applications

  • Implement additional validation controls

The antibody resources developed for Arabidopsis research provide valuable tools that, with proper validation, may extend to other plant species to facilitate comparative studies .

What controls are essential when using At1g59790 antibody in experimental procedures?

Implementing appropriate controls is critical for reliable data interpretation with At1g59790 antibody:

• Essential negative controls:

  • Primary antibody omission

  • Secondary antibody only

  • Pre-immune serum (if available)

  • Samples from knockout/knockdown mutants

• Critical positive controls:

  • Wild-type Arabidopsis samples

  • Recombinant At1g59790 protein (if available)

  • Overexpression lines

• Technical controls:

  • Loading controls (housekeeping proteins)

  • Transfer efficiency verification

  • Blocking efficiency assessment

• Validation controls:

  • Peptide competition assay

  • Antibody dilution series

  • Cross-reactivity testing

Research on Arabidopsis antibodies demonstrates that validation against respective mutant backgrounds provides the strongest confirmation of specificity .

How should quantitative data from At1g59790 antibody experiments be analyzed and interpreted?

For robust quantitative analysis of At1g59790 antibody data:

• Image acquisition considerations:

  • Capture images within linear dynamic range

  • Standardize exposure settings across experiments

  • Avoid oversaturation that compromises quantification

• Densitometric analysis:

  • Use specialized software (ImageJ, Image Lab)

  • Define consistent measurement parameters

  • Subtract background appropriately

  • Normalize to loading controls

• Statistical approaches:

  • Use minimum 3 biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Consider paired analyses for treatment comparisons

  • Report variability (standard deviation or standard error)

• Data presentation:

  • Include representative blot images

  • Present quantification in graphical format

  • Show all data points alongside means

  • Clearly indicate statistical significance

What methodological challenges exist when using At1g59790 antibody in complex plant tissues?

Working with At1g59790 antibody in complex plant tissues presents specific challenges requiring methodological solutions:

• Tissue heterogeneity issues:

  • Target protein may vary across cell types

  • Consider cell type-specific extraction methods

  • Implement microdissection for tissue-specific analysis

  • Compare whole-tissue vs. isolated cell populations

• Interfering compounds management:

  • Plant tissues contain pigments, phenolics, and secondary metabolites

  • Add PVPP or activated charcoal to extraction buffers

  • Include β-mercaptoethanol to prevent oxidation

  • Perform TCA/acetone precipitation to remove contaminants

• Protein accessibility challenges:

  • Cell wall structures may limit extraction efficiency

  • Optimize mechanical disruption methods

  • Test enzymatic pre-treatments

  • Consider alternative extraction buffers

• Background signal reduction:

  • Plant tissues often generate higher background

  • Increase blocking stringency

  • Perform additional washing steps

  • Test alternative blocking agents (fish gelatin, casein)

Research on Arabidopsis antibodies has shown that these methodological adaptations significantly improve detection success in complex plant tissues .