At3g59170 Antibody

What is At3g59170 Antibody and what organism does it target?

At3g59170 Antibody is a polyclonal antibody developed against recombinant Arabidopsis thaliana At3g59170 protein. It specifically recognizes and binds to the At3g59170 protein in Arabidopsis thaliana (Mouse-ear cress), making it a valuable tool for researchers studying this model plant organism. The antibody is raised in rabbits and purified using antigen affinity methods to ensure specificity and reduce background signal in experimental applications .

What are the primary applications for At3g59170 Antibody in plant research?

At3g59170 Antibody has been validated for use in Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications in plant research. These techniques allow researchers to detect and quantify the presence of the At3g59170 protein in various experimental contexts. For Western blots, the antibody helps identify the specific protein band at its expected molecular weight in protein extracts from Arabidopsis thaliana tissues. For ELISA applications, it enables quantitative measurement of the target protein in solution .

How should At3g59170 Antibody be stored and reconstituted for optimal performance?

For optimal performance and longevity, At3g59170 Antibody should be stored at -20°C or -80°C upon receipt. The antibody is typically provided in a stabilized liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. To minimize antibody degradation, avoid repeated freeze-thaw cycles by dividing the stock antibody into small aliquots after initial thawing. Before each use, briefly centrifuge the tube to collect any solution that might adhere to the cap or sides of the tube. This careful handling helps maintain antibody activity and specificity throughout your research project timeline .

How does At3g59170 Antibody compare to nanobody approaches for protein targeting in plant research?

While At3g59170 Antibody represents a traditional polyclonal antibody approach to protein targeting, recent advances in antibody technology have introduced nanobodies as an alternative system with distinct advantages in some research contexts. Traditional polyclonal antibodies like At3g59170 Antibody have a molecular weight of approximately 150 kDa and consist of two heavy and two light chains, whereas nanobodies (derived from camelid species such as alpacas) are significantly smaller (12-15 kDa) and consist of a single domain .

What cross-reactivity considerations should be evaluated when using At3g59170 Antibody in related plant species?

When extending research beyond Arabidopsis thaliana to related plant species, careful validation of At3g59170 Antibody cross-reactivity is essential. Though the antibody is specifically designed for Arabidopsis thaliana, sequence homology analysis of the At3g59170 protein across different plant species can predict potential cross-reactivity. Researchers should conduct preliminary Western blot analyses with protein extracts from the species of interest to confirm antibody specificity before performing comprehensive experiments.

Drawing from cross-reactivity patterns observed with other plant antibodies, such as the tubulin alpha chain antibody that successfully recognizes targets across diverse species including Chlamydomonas reinhardii and Zea mays, careful epitope analysis can help predict potential cross-reactivity of At3g59170 Antibody . When cross-reactivity is observed, titration experiments should be performed to optimize antibody concentration for maximum specific signal and minimum background in each new species.

How can computational approaches complement At3g59170 Antibody-based protein studies?

Integrating computational approaches with At3g59170 Antibody-based protein studies can significantly enhance research outcomes through multi-dimensional data analysis. Recent advancements in machine learning models for antibody-antigen binding prediction, as demonstrated in library-on-library approaches, offer powerful complementary tools for antibody-based research .

Researchers can leverage active learning strategies to predict antibody-antigen interactions with reduced experimental testing requirements, potentially decreasing the number of required antigen mutant variants by up to 35% . For At3g59170 protein studies, this computational approach can help predict structural interactions, optimize experimental design, and interpret complex binding patterns that might not be evident from antibody-based detection methods alone. This integrated approach is particularly valuable for characterizing novel protein-protein interactions involving At3g59170 or when mapping functional domains within the protein structure.

What controls are essential when using At3g59170 Antibody in Western blot experiments?

Implementing rigorous controls is critical for generating reliable and interpretable data when using At3g59170 Antibody in Western blot experiments. The following controls should be considered mandatory:

• Positive Control: Include protein extracts from wild-type Arabidopsis thaliana known to express At3g59170 protein.

• Negative Control: Use protein extracts from:

  • At3g59170 knockout/knockdown mutant lines

  • Non-plant tissue or unrelated organisms where the protein is absent

• Loading Control: Include detection of a constitutively expressed protein (e.g., actin or tubulin) to normalize protein loading across samples.

• Primary Antibody Controls:

  • Omission of primary antibody

  • Pre-immune serum at the same dilution as the antibody

  • Pre-absorption of the antibody with the immunizing peptide/protein

• Secondary Antibody Control: Incubation with secondary antibody alone to detect non-specific binding.

When analyzing results, compare signal strength and specificity across all controls to confirm that observed bands represent genuine At3g59170 protein detection rather than artifacts or cross-reactivity .

What is the optimal dilution range for At3g59170 Antibody in different experimental applications?

Determining the optimal dilution for At3g59170 Antibody varies by application and requires empirical optimization. Based on typical dilution ranges for similar polyclonal antibodies:

For Western blot applications specifically, perform a dilution series experiment including 1:500, 1:1000, 1:2000, and 1:5000 dilutions to identify the concentration that provides optimal specific signal with minimal background. The ideal dilution may vary depending on target protein abundance in your specific samples and the detection system used .

How should sample preparation be optimized for detecting At3g59170 protein in plant tissues?

Optimal sample preparation is crucial for successful detection of At3g59170 protein in plant tissues. The following methodological approach is recommended:

• Tissue Collection and Preservation:

  • Harvest plant tissues at appropriate developmental stages when At3g59170 expression is expected

  • Flash-freeze samples immediately in liquid nitrogen

  • Store at -80°C until processing to prevent protein degradation

• Extraction Buffer Optimization:

  • Use a buffer containing: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100

  • Include fresh protease inhibitors (complete cocktail)

  • Add phosphatase inhibitors if phosphorylation status is important

  • Consider adding 10 mM DTT to maintain reducing conditions

• Homogenization Technique:

  • Grind tissues thoroughly in liquid nitrogen using a mortar and pestle

  • Maintain cold temperature throughout the homogenization process

  • For recalcitrant tissues, consider mechanical disruption methods (bead-beating)

• Protein Solubilization and Clarification:

  • Incubate crude extracts at 4°C with gentle rotation for 30 minutes

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

  • Carefully collect the supernatant without disturbing the pellet

  • Perform a second centrifugation step to ensure complete removal of insoluble material

• Protein Quantification and Standardization:

  • Determine protein concentration using Bradford or BCA assay

  • Standardize all samples to equal protein concentration

  • Store aliquots at -80°C to avoid freeze-thaw cycles

This methodical approach ensures maximum protein recovery while minimizing degradation and modification, critical for accurate detection of At3g59170 protein .

What are common causes of false negative results when using At3g59170 Antibody and how can they be addressed?

False negative results when using At3g59170 Antibody can stem from multiple sources that researchers should systematically evaluate and address:

• Protein Degradation: At3g59170 protein may degrade during sample preparation.

  • Solution: Add fresh protease inhibitors to extraction buffers, maintain samples at 4°C during processing, and avoid freeze-thaw cycles.

• Insufficient Protein Loading: Target protein concentration may be below detection threshold.

  • Solution: Increase the amount of total protein loaded (50-100 µg) and verify with Ponceau S staining.

• Inadequate Transfer: Inefficient transfer of proteins to membrane.

  • Solution: Optimize transfer conditions (time, voltage, buffer composition) and confirm transfer efficiency with reversible total protein stains.

• Suboptimal Blocking: Excessive blocking may mask epitopes.

  • Solution: Test different blocking agents (BSA vs. non-fat milk) and concentrations (3-5%).

• Epitope Masking: Post-translational modifications or protein interactions may obscure the epitope.

  • Solution: Test denaturing conditions that may expose hidden epitopes, or try immunoprecipitation under native conditions followed by Western blotting.

• Antibody Activity Loss: The antibody may have lost activity during storage.

  • Solution: Use a fresh antibody aliquot and verify antibody activity with a positive control sample known to contain At3g59170 protein .

How can researchers distinguish between specific and non-specific bands when interpreting Western blot results with At3g59170 Antibody?

Distinguishing between specific and non-specific bands requires systematic analysis and multiple validation approaches:

• Molecular Weight Verification: The At3g59170 protein should appear at its predicted molecular weight. Any deviation should be investigated for potential post-translational modifications or processing.

• Knockout/Knockdown Validation: The most definitive approach is to compare wild-type samples with genetic knockout or knockdown lines. The specific band should be absent or reduced in intensity in these negative controls.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide before application to the blot. Specific bands should disappear while non-specific bands remain.

• Gradient Analysis: Examine how band intensity changes with increasing protein amounts. Specific bands typically show proportional increases with higher protein loading, while non-specific bands may not follow this pattern.

• Alternative Antibody Validation: If available, test a second antibody raised against a different epitope of the same protein. Specific bands should be detected by both antibodies.

• Subcellular Fractionation: If the subcellular localization of At3g59170 is known, analyze different cellular fractions. The specific band should be enriched in the appropriate fraction .

What approaches can address cross-reactivity issues with At3g59170 Antibody?

When facing cross-reactivity challenges with At3g59170 Antibody, researchers can employ several strategic approaches to improve specificity:

• Antibody Purification: Consider additional purification steps:

  • Perform affinity purification using the immobilized immunogen

  • Use negative selection against potential cross-reactive proteins

• Blocking Optimization: Modify blocking conditions:

  • Test various blocking agents (BSA, casein, commercial blocking solutions)

  • Increase blocking time or concentration

  • Add components to reduce non-specific interactions (0.1-0.5% Tween-20)

• Dilution Optimization: Higher dilutions often improve specificity:

  • Perform systematic titration experiments (1:1000, 1:2000, 1:5000)

  • Balance decreased cross-reactivity against reduced target signal

• Stringency Adjustment: Modify washing conditions:

  • Increase salt concentration in wash buffers (from 150 mM to 300-500 mM NaCl)

  • Add detergents (0.1-0.5% SDS) to wash buffers

  • Extend washing time or add additional wash steps

• Alternative Detection Systems: Consider different detection methods:

  • Switch from colorimetric to chemiluminescence detection for improved signal-to-noise ratio

  • Use highly cross-adsorbed secondary antibodies

  • Consider two-color Western blot systems to differentiate target from cross-reactive proteins

• Immunoprecipitation-Western Blot: Perform immunoprecipitation before Western blotting to enrich for the specific target and reduce potential cross-reactive proteins .

How might At3g59170 Antibody be integrated into multi-antibody experiments for protein complex studies?

Integrating At3g59170 Antibody into multi-antibody experiments for protein complex studies requires careful planning and methodological considerations:

• Co-Immunoprecipitation (Co-IP) Approaches:

  • Use At3g59170 Antibody as the primary precipitation antibody to pull down the target protein along with its interaction partners

  • Alternatively, use antibodies against suspected interaction partners for precipitation and detect At3g59170 in the immunoprecipitate

  • Consider crosslinking approaches to stabilize transient interactions before immunoprecipitation

• Sequential Immunoprecipitation:

  • Perform tandem immunoprecipitation using At3g59170 Antibody followed by antibodies against potential interaction partners

  • This approach increases specificity by requiring both proteins to be present in the same complex

• Proximity Ligation Assays (PLA):

  • Combine At3g59170 Antibody with antibodies against potential interaction partners

  • PLA signals are generated only when the two antibodies are in close proximity (30-40 nm)

  • This approach provides spatial information about protein interactions in situ

• Multiple Immunofluorescence Labeling:

  • Use At3g59170 Antibody in combination with antibodies against other proteins

  • Select secondary antibodies with distinct fluorophores to allow simultaneous visualization

  • Include appropriate controls to ensure no cross-reactivity between antibodies

• Antibody Compatibility Considerations:

  • Ensure antibodies are raised in different host species to allow specific secondary antibody detection

  • If antibodies are from the same species, consider direct labeling with different fluorophores

  • Verify that antibody combinations do not interfere with each other's binding .

What considerations are important when designing time-course experiments with At3g59170 Antibody?

Designing effective time-course experiments with At3g59170 Antibody requires attention to several key methodological considerations:

• Temporal Sampling Strategy:

  • Establish appropriate time points based on the biological process being studied

  • Include early time points to capture rapid responses and later points for delayed effects

  • Consider logarithmic rather than linear time sampling for processes with rapid initial changes

• Sample Preparation Consistency:

  • Process all time points identically to minimize technical variation

  • Consider preparing all samples simultaneously and freezing aliquots for later analysis

  • If processing in batches is necessary, include reference samples across batches

• Quantification and Normalization:

  • Use densitometry for Western blot quantification with appropriate software

  • Normalize to loading controls that remain stable throughout the time course

  • Consider multiple normalization controls (housekeeping proteins, total protein stains)

• Controls for Temporal Stability:

  • Include controls to assess protein stability under experimental conditions

  • Monitor potential degradation of the target protein over time

  • Include appropriate negative controls at each time point

• Data Representation:

  • Present data as fold-change relative to baseline (time zero) or appropriate control

  • Include statistical analysis across biological replicates

  • Consider regression analysis for continuous time-dependent changes

• Experimental Design Table:

This systematic approach ensures detection of genuine temporal changes in At3g59170 protein levels while minimizing experimental artifacts .

How can researchers develop custom modifications to standard protocols for optimizing At3g59170 Antibody performance in challenging samples?

Developing custom protocol modifications for challenging samples requires systematic troubleshooting and optimization:

• Extraction Buffer Customization:

  • For samples with high phenolic content: Add polyvinylpolypyrrolidone (PVPP, 2-4%) to bind phenolics

  • For samples with abundant interfering compounds: Include activated charcoal (0.5-1%) during extraction

  • For tissues with high proteolytic activity: Double the concentration of protease inhibitors and add EDTA (5 mM)

• Membrane Selection and Treatment:

  • Test different membrane types: PVDF offers higher protein binding capacity than nitrocellulose

  • Optimize pore size: 0.2 μm pores for small proteins, 0.45 μm for larger proteins

  • Consider enhanced chemiluminescence (ECL) compatible membranes for improved sensitivity

• Signal Enhancement Strategies:

  • Employ signal amplification systems like tyramide signal amplification (TSA)

  • Use biotin-streptavidin systems for enhanced detection sensitivity

  • Consider polymer-based detection systems that provide higher sensitivity than traditional methods

• Sample Pre-treatment Options:

  • For complex plant tissues: Implement fractionation to enrich for subcellular compartments

  • For samples with high lipid content: Include additional delipidation steps (acetone precipitation)

  • For heavily glycosylated samples: Consider deglycosylation treatments if glycans might mask epitopes

• Antigen Retrieval Techniques:

  • Adapt immunohistochemistry antigen retrieval methods:

    • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

    • Enzymatic treatment with proteases to expose hidden epitopes

    • Chemical treatments (SDS, urea) to partially denature proteins and expose epitopes

This methodical approach to protocol customization enables researchers to overcome sample-specific challenges while maintaining the specificity and sensitivity of At3g59170 Antibody detection .