At3g05170 Antibody

What is At3g05170 Antibody and what organism does it target?

At3g05170 Antibody (product code CSB-PA943266XA01DOA) is a polyclonal antibody raised against recombinant Arabidopsis thaliana At3g05170 protein. It specifically recognizes and binds to the At3g05170 protein in Arabidopsis thaliana (Mouse-ear cress), making it a valuable tool for studying protein expression in this model plant organism. The antibody is generated in rabbits and purified using antigen affinity methods to ensure specificity to the target protein .

What are the validated applications for At3g05170 Antibody?

The At3g05170 Antibody has been validated for use in Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications. These techniques allow researchers to detect and quantify the At3g05170 protein in complex biological samples. The antibody's specificity ensures proper identification of the antigen in these applications, providing reliable results for research purposes .

What are the optimal storage conditions for At3g05170 Antibody?

For optimal preservation of antibody activity, At3g05170 Antibody should be stored at either -20°C or -80°C upon receipt. It's crucial to avoid repeated freeze-thaw cycles as these can compromise antibody function. The antibody is supplied in liquid form in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. This formulation helps maintain antibody stability during storage while preventing microbial contamination .

What approaches can be used to verify At3g05170 Antibody specificity in Arabidopsis thaliana samples?

Verifying antibody specificity is crucial for reliable experimental outcomes. For At3g05170 Antibody, researchers can employ several validation strategies:

• Peptide competition assays - Pre-incubating the antibody with excess recombinant At3g05170 protein before application to samples

• Using At3g05170 knockout/knockdown lines as negative controls

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Comparing reactivity patterns across different tissue types with known expression profiles

These approaches help establish confidence in antibody specificity, similar to specificity verification methods described for other research antibodies .

How can At3g05170 Antibody be integrated into genotype-phenotype studies?

At3g05170 Antibody can serve as a valuable tool in genotype-phenotype correlation studies by enabling protein-level analysis of At3g05170 expression across different Arabidopsis thaliana genotypes. Researchers can use this antibody in conjunction with NGS (Next-Generation Sequencing) data to link genetic variations to protein expression patterns. This integrated approach allows for functional validation of genetic findings and provides insights into how genetic variations affect protein abundance and function. Similar to the genotype-phenotype linked antibody screening approaches, researchers can design experiments that combine genetic analysis with protein-level detection using this antibody .

What is the recommended protocol for using At3g05170 Antibody in Western Blotting?

For optimal Western Blotting results with At3g05170 Antibody, follow this methodological approach:

• Sample preparation:

  • Extract proteins from Arabidopsis thaliana tissues using appropriate lysis buffer

  • Determine protein concentration using Bradford or BCA assay

  • Prepare samples containing 20-50 μg total protein with reducing sample buffer

• SDS-PAGE and transfer:

  • Separate proteins using 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.45 μm pore size)

• Blocking and antibody incubation:

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

  • Incubate with At3g05170 Antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C

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

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Detection:

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

  • Develop using ECL substrate and visualize using film or digital imaging system

This protocol is based on standard Western blotting procedures for polyclonal antibodies with considerations for the specific properties of At3g05170 Antibody .

How should At3g05170 Antibody be optimized for ELISA applications?

For developing a robust ELISA assay using At3g05170 Antibody, consider this methodological workflow:

Begin with these conditions and perform checkerboard titrations to determine optimal antibody and sample concentrations for your specific application. This approach is similar to optimization strategies used for other research antibodies in ELISA formats .

What considerations are important when using At3g05170 Antibody in complex plant tissue samples?

When working with complex plant tissue samples, several methodological considerations are important:

• Sample preparation:

  • Optimize extraction buffers to efficiently solubilize At3g05170 protein while minimizing interference from plant-specific compounds

  • Consider including protease inhibitors to prevent target degradation

  • Include reducing agents if the target protein contains disulfide bonds

• Background reduction:

  • Pre-clear lysates by centrifugation to remove insoluble material

  • Consider pre-adsorption of the antibody with wild-type plant extracts from tissues not expressing the target

  • Optimize blocking conditions to reduce non-specific binding to plant components

• Controls:

  • Include extracts from tissues with known expression patterns

  • When possible, use knockout/knockdown lines as negative controls

• Detection optimization:

  • Adjust antibody concentration based on target abundance

  • Consider enhanced chemiluminescence substrates for low-abundance targets

These considerations help overcome the challenges associated with plant-specific matrix effects and ensure specific detection of At3g05170 protein .

How can researchers address weak or absent signal when using At3g05170 Antibody?

When encountering weak or absent signals with At3g05170 Antibody, consider the following systematic troubleshooting approach:

• Antibody activity:

  • Verify antibody integrity by avoiding repeated freeze-thaw cycles

  • Check expiration date and storage conditions

  • Consider using a positive control sample with known target expression

• Protocol optimization:

  • Increase antibody concentration (try 2-5x higher concentration)

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

  • Reduce washing stringency

  • Try different detection systems with higher sensitivity

• Sample-related issues:

  • Ensure proper sample preparation to expose epitopes

  • Verify protein transfer efficiency in Western blotting

  • Confirm target protein expression in your experimental conditions

  • Consider target protein degradation during preparation

• Expression levels:

  • Use techniques to enrich for the target protein (immunoprecipitation)

  • Consider developmental stage or experimental conditions that might affect expression

This methodical approach helps identify the source of signal problems and guides appropriate interventions .

What strategies can address non-specific binding or high background with At3g05170 Antibody?

When facing high background or non-specific binding issues, implement these research-proven strategies:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time or concentration

  • Add 0.1-0.3% Tween-20 to washing and antibody dilution buffers

• Antibody dilution:

  • Increase antibody dilution to reduce non-specific binding

  • Prepare antibody dilutions in fresh blocking buffer

  • Consider shorter incubation times at higher antibody concentrations

• Sample preparation:

  • Include additional washing steps

  • Perform pre-clearing of lysates

  • Add 0.1-0.5M NaCl to washing buffers to increase stringency

• Cross-reactivity reduction:

  • Pre-absorb antibody with proteins from non-target species

  • Purify antibody using affinity chromatography

  • Use monoclonal antibodies if available for the target

These approaches systematically address the common causes of background and non-specific binding in immunoassays .

How can researchers quantitatively analyze At3g05170 protein expression data?

For quantitative analysis of At3g05170 protein expression data, implement these methodological approaches:

• Western blot quantification:

  • Use housekeeping proteins (e.g., actin, tubulin) as loading controls

  • Employ densitometry software to measure band intensity

  • Establish a standard curve using recombinant At3g05170 protein

  • Normalize target protein signal to loading control

• ELISA quantification:

  • Generate standard curves using purified recombinant At3g05170 protein

  • Ensure samples fall within the linear range of detection

  • Run samples in technical triplicates

  • Apply appropriate statistical analysis (t-tests, ANOVA) to compare groups

• Data normalization strategies:

  • Account for total protein concentration

  • Consider tissue-specific reference proteins

  • Apply log transformation for wide-ranging expression data

• Software tools:

  • ImageJ for Western blot analysis

  • GraphPad Prism or R for statistical analysis and visualization

  • Consider using specialized ELISA analysis software for plate reader data

These quantitative approaches provide robust, reproducible analysis of protein expression data for publishing-quality results .

How can At3g05170 Antibody be utilized in multiparameter analysis approaches?

At3g05170 Antibody can be integrated into multiparameter analysis workflows to provide comprehensive insights into protein function and interactions:

• Co-immunoprecipitation studies:

  • Use At3g05170 Antibody to pull down the target protein and associated complexes

  • Analyze precipitated proteins by mass spectrometry to identify interaction partners

  • Verify interactions using reciprocal co-IP with antibodies against putative partners

• Chromatin immunoprecipitation (if At3g05170 has DNA-binding functions):

  • Optimize chromatin fragmentation and IP conditions

  • Use At3g05170 Antibody to isolate protein-DNA complexes

  • Sequence precipitated DNA to identify binding sites

• Multiplexed immunoassays:

  • Combine At3g05170 Antibody with antibodies against other proteins in the same pathway

  • Develop multiplex Western blotting using different visualization methods

  • Implement multiplex ELISA for simultaneous quantification of multiple proteins

These approaches parallel advanced antibody screening techniques that link genotype and phenotype, as described in antibody development research .

What considerations are important when interpreting contradictory results with At3g05170 Antibody?

When faced with contradictory results using At3g05170 Antibody, researchers should implement this analytical framework:

• Technical variability assessment:

  • Evaluate reproducibility across technical replicates

  • Consider batch effects from antibody lots or reagent preparations

  • Verify assay performance using appropriate controls

• Biological variability analysis:

  • Assess natural variation in At3g05170 expression across growth conditions

  • Consider post-translational modifications that might affect antibody recognition

  • Evaluate developmental stage-specific expression patterns

• Methodological differences:

  • Compare results across different detection methods (Western blot vs. ELISA)

  • Consider epitope accessibility in different sample preparation methods

  • Evaluate the impact of different buffers or reagents on antibody performance

• Verification strategies:

  • Use alternative detection methods (RT-PCR for mRNA, mass spectrometry for protein)

  • Consider orthogonal antibodies recognizing different epitopes

  • Implement genetic approaches (knockout/knockdown) to validate findings