At5g36820 Antibody

What is At5g36820 and why is it significant in plant research?

At5g36820 is a gene in Arabidopsis thaliana that encodes a protein with the UniProt accession number P0DI01. The significance of this gene in plant research stems from its role in Arabidopsis cellular processes. Researchers use antibodies against this protein to investigate its expression patterns, localization, and functional roles in plant development and stress responses. The antibody enables visualization of protein expression across different tissues and under varying experimental conditions, providing insights into regulatory networks and protein-protein interactions in this model plant organism .

What are the optimal storage conditions for At5g36820 antibody?

The optimal storage conditions for At5g36820 antibody are -20°C or -80°C upon receipt. It is crucial to avoid repeated freeze-thaw cycles as these can compromise antibody functionality and binding efficacy. The antibody is typically supplied in liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For short-term usage (1-2 weeks), storing small aliquots at 4°C can maintain functionality while minimizing freeze-thaw damage. Researchers should document the number of freeze-thaw cycles and validate antibody performance after extended storage periods through positive control experiments.

What applications is the At5g36820 antibody validated for?

The At5g36820 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . These techniques allow researchers to detect and quantify the target protein in plant tissue samples. While these are the specifically validated applications, researchers often adapt antibodies for additional techniques:

*Specific dilutions should be optimized for each experimental setup and tissue type.

How can I assess potential cross-reactivity of At5g36820 antibody with other plant species?

Assessing cross-reactivity of the At5g36820 antibody with other plant species requires a systematic approach. First, conduct sequence homology analysis of the At5g36820 protein across species of interest using bioinformatics tools like BLAST to identify proteins with similar epitopes. Proteins with >70% sequence identity in the epitope region have higher likelihood of cross-reactivity. Second, perform Western blot validation using protein extracts from multiple species alongside Arabidopsis thaliana as a positive control .

For rigorous validation, implement the following protocol:

• Extract proteins from target species using identical protocols

• Load equal amounts of protein (15-30 μg) per lane

• Include recombinant At5g36820 protein as a positive control

• Perform Western blot with a dilution series (1:500, 1:1000, 1:2000)

• Document band patterns and intensities across species

• Confirm specificity through immunoprecipitation followed by mass spectrometry

This methodological approach provides quantitative evidence of cross-reactivity and helps determine appropriate applications in comparative plant biology research .

What strategies can improve signal-to-noise ratio when using At5g36820 antibody in low-expression tissues?

Improving signal-to-noise ratio when working with At5g36820 antibody in tissues with low target protein expression requires optimization of several experimental parameters. First, consider protein enrichment techniques such as immunoprecipitation or subcellular fractionation to concentrate the target protein before analysis. Second, optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) and determining which minimizes background without reducing specific signals .

Additional methodological improvements include:

• Extended primary antibody incubation (overnight at 4°C) to maximize binding to low-abundance targets

• Implementing signal amplification systems such as biotin-streptavidin or tyramide signal amplification

• Using highly sensitive detection reagents (enhanced chemiluminescence or fluorescent secondary antibodies)

• Optimizing membrane washing protocols (increased number of washes, higher detergent concentration)

• Testing different secondary antibodies to find optimal signal-to-noise characteristics

For Western blots specifically, loading higher protein amounts (50-100 μg) and using gradient gels to improve separation can significantly enhance detection of low-abundance targets. Quantitative comparison of different optimization strategies shows that signal amplification methods can improve detection limits by 5-10 fold compared to standard protocols .

How do experimental conditions affect epitope accessibility of At5g36820 protein?

Epitope accessibility of At5g36820 protein can be significantly influenced by experimental conditions, affecting antibody binding efficiency and experimental outcomes. The structural conformation of proteins depends on multiple factors including sample preparation methods, buffer compositions, and detection techniques. For the At5g36820 antibody, which recognizes a specific epitope of the recombinant protein, accessibility may be compromised under denaturing conditions that alter the protein's native structure .

Key experimental factors affecting epitope accessibility include:

• Fixation methods: Crosslinking fixatives (e.g., formaldehyde) can mask epitopes while precipitating fixatives (e.g., acetone) may preserve epitope accessibility

• Protein extraction buffers: Ionic strength, pH, and detergent types influence protein conformation

• Reducing conditions: DTT or β-mercaptoethanol can disrupt disulfide bonds, altering epitope structure

• Heat treatment: Excessive heating during sample preparation may cause irreversible protein denaturation

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications may obscure the epitope

To optimize epitope accessibility, implement antigen retrieval methods such as heat-induced epitope retrieval (HIER) or proteolytic-induced epitope retrieval (PIER) for fixed samples. For native protein detection, gentle extraction procedures using non-ionic detergents at physiological pH help maintain protein conformation and epitope integrity .

What is the optimal validation protocol for confirming antibody specificity in new experimental systems?

Validating At5g36820 antibody specificity in new experimental systems requires a multi-step approach to ensure reliable results. A comprehensive validation protocol should include positive and negative controls, along with multiple orthogonal techniques to confirm target specificity .

The recommended validation workflow includes:

• Western blot analysis using:

  • Wild-type Arabidopsis thaliana extracts (positive control)

  • At5g36820 knockout/knockdown lines (negative control)

  • Recombinant At5g36820 protein (positive control)

  • Pre-absorption test with immunizing peptide

• Immunoprecipitation followed by mass spectrometry:

  • Confirm pulled-down proteins match At5g36820

  • Quantify enrichment relative to IgG control

• Genetic validation approaches:

  • Test antibody reactivity in plants with CRISPR-edited At5g36820

  • Examine correlation between transcript levels (RT-qPCR) and protein detection

• Dilution series and titration:

  • Determine optimal antibody concentration

  • Document signal reduction with decreasing target concentration

This methodological framework provides multiple lines of evidence for antibody specificity, crucial for publication-quality research. Documentation of all validation steps should be maintained and reported in publications to ensure reproducibility .

How should sample preparation be optimized for detecting At5g36820 in different plant tissues?

Optimizing sample preparation for detecting At5g36820 across different plant tissues requires tissue-specific approaches to overcome varying matrices, protein abundances, and potential interfering compounds. The extraction protocol must effectively solubilize the target protein while preserving epitope integrity .

Tissue-specific optimization strategies include:

For all tissue types, incorporate these methodological considerations:

• Perform extraction at 4°C to minimize proteolysis

• Include protease inhibitor cocktail freshly added before extraction

• Clarify extracts by high-speed centrifugation (20,000 × g for 15 minutes)

• Quantify protein concentration using Bradford or BCA assay prior to immunoblotting

• Normalize loading based on total protein rather than housekeeping genes for cross-tissue comparisons

These tissue-specific approaches maximize protein recovery while minimizing interference from tissue-specific compounds that could affect antibody binding or generate background signals .

What detection systems provide optimal sensitivity for At5g36820 antibody in various applications?

Selecting the optimal detection system for At5g36820 antibody depends on the application, required sensitivity, and available instrumentation. Different detection systems offer varying levels of sensitivity, dynamic range, and compatibility with downstream analyses .

Comparative analysis of detection systems:

For Western blot applications using At5g36820 antibody, enhanced chemiluminescence (ECL) provides good sensitivity with reasonable cost and equipment requirements. For quantitative analysis, fluorescent secondary antibodies offer superior linearity and multiplexing capability, allowing simultaneous detection of At5g36820 alongside loading controls or other proteins of interest.

Implementation considerations include:

• Match detection system to expected target abundance

• For quantitative applications, fluorescent systems offer better linearity

• For ultra-sensitive detection, consider signal amplification systems

• Validate detection limits using dilution series of recombinant protein

• Optimize exposure times to prevent signal saturation in high-expression samples

The methodological choice of detection system should be guided by experimental objectives and reported in publications to ensure reproducibility .

How can I quantitatively analyze At5g36820 expression across different experimental conditions?

Quantitative analysis of At5g36820 expression across experimental conditions requires rigorous methodological approaches to ensure accurate and reproducible results. For Western blot analysis, densitometry combined with appropriate normalization is the standard approach .

Implement this methodological workflow:

• Experimental design considerations:

  • Include biological replicates (minimum n=3)

  • Process all samples simultaneously when possible

  • Include internal reference standards on each blot

• Image acquisition parameters:

  • Capture images within linear detection range

  • Use same exposure settings across replicates

  • Document image acquisition parameters

• Quantification procedure:

  • Use scientific image analysis software (ImageJ, Image Lab, etc.)

  • Define consistent region of interest for each band

  • Subtract local background from each measurement

  • Normalize to total protein stain (SYPRO Ruby, Ponceau S) rather than single housekeeping proteins

  • Calculate relative expression using consistent reference point

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Assess significance using p-value thresholds

  • Report both fold-change and statistical significance

• Data presentation:

  • Display normalized intensities with error bars

  • Include representative blot images

  • Report both raw and normalized values in data tables

This standardized quantification approach minimizes technical variability and provides statistically robust measurements of At5g36820 expression changes across experimental conditions .

What are the common sources of false positive and false negative results when using At5g36820 antibody?

Understanding potential sources of false results is critical for accurate interpretation of experiments using At5g36820 antibody. Both false positives and false negatives can arise from multiple technical and biological factors .

Common sources of false positive results include:

• Cross-reactivity with structurally similar proteins, particularly in non-Arabidopsis species

• Non-specific binding to high-abundance proteins in plant extracts

• Secondary antibody binding to endogenous plant immunoglobulins

• Matrix effects from plant compounds interacting with detection reagents

• Sample contamination with recombinant protein or positive control materials

Common sources of false negative results include:

• Epitope masking due to protein modifications or fixation procedures

• Target protein degradation during sample preparation

• Insufficient antibody concentration or incubation time

• Inefficient protein transfer in Western blotting

• Inhibitory compounds in plant extracts interfering with antibody binding

Methodological approaches to mitigate false results:

Implementing these methodological controls and documenting experimental conditions thoroughly helps distinguish true signals from artifacts, ensuring reliable research outcomes .

How do post-translational modifications affect At5g36820 detection and quantification?

Post-translational modifications (PTMs) of At5g36820 protein can significantly impact antibody detection and quantification by altering epitope accessibility, protein migration patterns, and antibody binding affinity. Understanding these effects is crucial for accurate interpretation of experimental results .

The impact of common PTMs on At5g36820 detection:

• Phosphorylation:

  • Can cause band shifts in SDS-PAGE (typically 1-5 kDa higher)

  • May enhance or inhibit antibody binding depending on epitope location

  • Reversible with phosphatase treatment prior to analysis

• Glycosylation:

  • Results in higher molecular weight bands (often diffuse pattern)

  • May mask epitopes in heavily glycosylated regions

  • Can be assessed using deglycosylation enzymes (PNGase F, O-glycosidase)

• Ubiquitination:

  • Creates higher molecular weight laddering pattern

  • May indicate protein targeted for degradation

  • Can be confirmed with co-immunoprecipitation using ubiquitin antibodies

• Proteolytic processing:

  • Generates fragments smaller than full-length protein

  • May result in loss of epitope in processed fragments

  • Preventable with appropriate protease inhibitors during extraction

Methodological approaches to assess PTM impacts:

• Enzymatic treatment comparisons:

  • Treat samples with phosphatases, deglycosylation enzymes, or deubiquitinating enzymes

  • Compare migration patterns and signal intensities before and after treatment

• 2D gel electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Visualize multiple isoforms of At5g36820 differing in PTMs

• Epitope mapping:

  • Use recombinant protein fragments to identify the specific region recognized by the antibody

  • Assess whether known PTM sites overlap with the epitope region

• Mass spectrometry validation:

  • Immunoprecipitate At5g36820 and analyze by mass spectrometry

  • Identify specific PTMs and their relative abundances

Understanding how PTMs affect At5g36820 detection enables more accurate interpretation of experimental results and can provide insights into the protein's regulation and function under different conditions .

What are the most effective troubleshooting strategies for weak or absent At5g36820 signals?

When encountering weak or absent signals when using the At5g36820 antibody, a systematic troubleshooting approach helps identify and resolve technical issues. The methodological troubleshooting workflow should progress from sample preparation to detection optimization .

Comprehensive troubleshooting strategy:

• Sample preparation assessment:

  • Verify protein extraction efficiency with total protein stain

  • Check for protein degradation using fresh samples with additional protease inhibitors

  • Confirm protein loading amounts (consider increasing for low-abundance targets)

  • Test alternate extraction buffers optimized for membrane proteins

• Primary antibody optimization:

  • Titrate antibody concentration (try 2-5× higher concentration)

  • Extend incubation time (overnight at 4°C)

  • Reduce washing stringency (lower detergent concentration)

  • Test different antibody dilution buffers (TBS-T with 1-5% BSA or milk)

• Detection system enhancement:

  • Switch to more sensitive detection system (ECL-Plus, fluorescent)

  • Extend exposure times for chemiluminescent detection

  • Implement signal amplification system (biotin-streptavidin)

  • Confirm secondary antibody compatibility and activity

• Protocol modifications:

  • For Western blots: optimize transfer conditions and membrane type

  • For ELISA: adjust coating buffer pH and blocking conditions

  • For all applications: reduce background with additional blocking and washes

• Positive control validation:

  • Run parallel experiment with recombinant At5g36820 protein

  • Test antibody functionality with known positive sample

  • Verify target is expressed in tissue type being studied

This methodical approach systematically eliminates potential sources of signal failure and provides documentation of optimization efforts for reproducible protocols .

How can researchers distinguish between specific and non-specific binding when using At5g36820 antibody?

Distinguishing between specific and non-specific binding is critical for accurate interpretation of results when using At5g36820 antibody. Multiple methodological approaches can be implemented to validate signal specificity .

Methodological approaches to confirm signal specificity:

• Knockout/knockdown validation:

  • Compare wild-type and At5g36820 knockout/knockdown plants

  • Specific signals should be absent or significantly reduced in knockout samples

  • Persistent bands in knockout samples indicate non-specific binding

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be eliminated in peptide-blocked samples

• Molecular weight verification:

  • Compare observed band size with predicted molecular weight of At5g36820

  • Consider post-translational modifications that may alter migration

  • Verify with recombinant protein standard

• Orthogonal detection methods:

  • Confirm results using independent antibody targeting different epitope

  • Correlate protein detection with mRNA expression data

  • Perform mass spectrometry validation of immunoprecipitated samples

• Technical controls:

  • Secondary antibody only control (omit primary antibody)

  • Isotype control (irrelevant primary antibody of same isotype)

  • Gradient of antigen concentration to demonstrate signal proportionality