At5g35200 Antibody

What is At5g35200 and why is it significant in Arabidopsis thaliana research?

At5g35200 (UniProt: Q9LHS0) is a gene locus in Arabidopsis thaliana (Mouse-ear cress) encoding a protein that has gained research interest in plant molecular biology. The antibody targeting this protein (product code CSB-PA881731XA01DOA) allows for specific detection in experimental systems. Similar to other plant protein antibodies, the significance lies in enabling researchers to track protein expression, localization, and interactions in both normal development and under various stress conditions . Antibodies like this serve as critical tools for understanding protein function in plant cellular processes, comparable to how ATG protein antibodies have advanced our understanding of autophagy mechanisms in plants .

What experimental applications are supported by At5g35200 antibody?

At5g35200 antibody is primarily designed for Western blot applications, consistent with other plant protein antibodies in the field. While specific applications for this particular antibody are not extensively documented, the antibody format and preparation suggest potential utility across several experimental approaches:

For reliable results, researchers should conduct preliminary validation experiments before applying this antibody to specialized techniques beyond Western blotting .

How should At5g35200 antibody be reconstituted and stored for optimal activity?

For maximum longevity and consistent experimental results, the lyophilized At5g35200 antibody should be reconstituted by adding 50 μl of sterile water, which aligns with standard practices for polyclonal antibodies in plant research. The reconstituted antibody should be stored at -20°C, and researchers are advised to create smaller working aliquots to prevent repeated freeze-thaw cycles that can significantly degrade antibody quality .

Research indicates that antibody degradation follows first-order kinetics, with activity decreasing by approximately 5-10% with each freeze-thaw cycle. For long-term experiments spanning several months, consider adding preservatives such as sodium azide (0.02%) or ProClin to working aliquots - though this option should be specifically requested when ordering the antibody .

What controls are essential when using At5g35200 antibody in experimental workflows?

Implementing appropriate controls is critical for accurate interpretation of results with plant-specific antibodies. For At5g35200 antibody experiments, the following controls should be considered:

• Positive control: Recombinant At5g35200 protein or extracts from wild-type Arabidopsis thaliana tissues known to express the target protein

• Negative control: Extracts from knockout mutants lacking At5g35200 expression, if available

• Secondary antibody-only control: To assess non-specific binding of the secondary detection system

• Pre-immune serum control: To establish baseline reactivity

• Cross-reactivity assessment: Testing reactivity against related proteins to confirm specificity

Researchers should note that this antibody has been confirmed to recognize recombinant protein but reactivity on endogenous protein requires further validation, similar to verification protocols used for other plant antibodies .

What is the optimal protein extraction protocol for At5g35200 detection in Arabidopsis tissues?

For effective At5g35200 protein extraction from Arabidopsis thaliana tissues, a modified protocol based on successful approaches for other plant proteins is recommended:

• Harvest fresh tissue (100-200 mg) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder using a pre-chilled mortar and pestle

• Add 500 μl extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA)

• Include freshly prepared protease inhibitor cocktail to prevent degradation

• Homogenize thoroughly and incubate on ice for 30 minutes with intermittent mixing

• Centrifuge at 13,000 × g for 15 minutes at 4°C

• Collect the supernatant containing soluble proteins

• Quantify protein concentration using Bradford or BCA assay

This protocol minimizes protein degradation while maximizing extraction efficiency, particularly important for membrane-associated or low-abundance proteins that might include At5g35200 .

How can immunolocalization studies with At5g35200 antibody be optimized for confocal microscopy?

While At5g35200 antibody has not been specifically validated for immunolocalization, researchers can adapt protocols used successfully with other plant antibodies:

• Tissue fixation: Use 4% paraformaldehyde in PBS for 2 hours at room temperature or overnight at 4°C

• Permeabilization: Treat with 0.1-0.5% Triton X-100 for 15-30 minutes to improve antibody access

• Antigen retrieval: Consider microwave-assisted citrate buffer (pH 6.0) treatment if initial attempts yield weak signals

• Blocking: Use 3-5% BSA or normal serum from the same species as the secondary antibody

• Primary antibody incubation: Test multiple dilutions (1:100 to 1:500) of At5g35200 antibody

• Secondary antibody selection: Choose fluorophore-conjugated secondary antibodies compatible with your microscopy setup

• Autofluorescence controls: Include unstained samples to account for plant tissue autofluorescence

• Co-localization markers: Consider using established organelle markers to determine subcellular localization

By systematically optimizing these parameters, researchers can establish reliable immunolocalization protocols for At5g35200 protein .

How can At5g35200 antibody be used to investigate protein-protein interactions?

To investigate protein-protein interactions involving At5g35200, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare protein extracts under non-denaturing conditions

  • Incubate with At5g35200 antibody pre-bound to Protein A/G beads

  • Wash extensively to remove non-specific interactions

  • Elute bound proteins and analyze by Western blot or mass spectrometry

  • Validate interactions using reciprocal Co-IP with antibodies against candidate interacting proteins

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant cells or tissues

  • Incubate with At5g35200 antibody and antibody against candidate interacting protein

  • Apply species-specific PLA probes with attached DNA oligonucleotides

  • Perform rolling circle amplification and detection with fluorescent probes

  • Analyze using confocal microscopy to visualize interaction sites in situ

These approaches can reveal both stable and transient interactions, providing insight into At5g35200's functional networks in plant cellular processes .

What strategies can resolve epitope masking issues when At5g35200 antibody shows inconsistent results?

Epitope masking is a common challenge in plant protein studies, particularly when target proteins form complexes or undergo post-translational modifications. For At5g35200 antibody, consider these advanced troubleshooting strategies:

• Denaturing conditions: Use stronger denaturing buffers containing 6-8M urea or 2% SDS to expose masked epitopes

• Chemical crosslinking: Preserve protein-protein interactions with formaldehyde or DSP before extraction

• Sequential extraction: Perform fractionated extraction with increasing detergent strengths

• Heat-induced epitope retrieval: For fixed tissues, heat samples in citrate buffer (pH 6.0) at 95°C for 10-20 minutes

• Protease treatment: Limited digestion with trypsin or proteinase K can expose hidden epitopes

• Native versus reducing conditions: Compare results under reducing (with DTT/β-mercaptoethanol) and non-reducing conditions

Systematic testing of these approaches can help identify optimal conditions for consistent At5g35200 detection across different experimental contexts .

How does the specificity of At5g35200 antibody compare to other plant protein antibodies?

When evaluating antibody specificity for plant research, comparative analysis provides valuable context. For At5g35200 antibody:

The specificity validation status of At5g35200 antibody is comparable to other plant antibodies in early research phases, where recombinant protein recognition has been established but endogenous protein detection requires further confirmation .

What approach should be taken when At5g35200 antibody shows unexpected cross-reactivity with related proteins?

Cross-reactivity is a significant concern in plant antibody applications, particularly with polyclonal antibodies like At5g35200 antibody. When unexpected cross-reactivity occurs:

• Epitope analysis: Compare the immunizing sequence with related proteins using bioinformatics tools to identify regions of homology

• Absorption controls: Pre-incubate the antibody with excess recombinant protein to block specific binding sites

• Gradient dilution testing: Test multiple antibody dilutions to find an optimal concentration that maximizes specific signal while minimizing cross-reactivity

• Knockout/knockdown validation: Compare results between wild-type and genetic lines with reduced or eliminated At5g35200 expression

• Peptide competition: Use synthetic peptides corresponding to the immunogen to block specific antibody binding

• Western blot optimization: Increase washing stringency and blocking concentration

These approaches can help distinguish true signals from cross-reactivity artifacts, enhancing experimental rigor when working with plant antibodies in complex biological systems .

How should quantitative data from At5g35200 antibody experiments be normalized for meaningful comparisons?

For rigorous quantitative analysis using At5g35200 antibody, proper normalization is essential:

• Loading control selection: For Western blots, use plant-specific housekeeping proteins like actin, tubulin, or GAPDH that remain stable under your experimental conditions

• Total protein normalization: Consider Ponceau S or Coomassie staining of membranes as an alternative to single protein loading controls

• Technical replicates: Perform at least three technical replicates to account for antibody binding variability

• Biological replicates: Include multiple independent biological samples (minimum n=3) for statistical validity

• Standard curve incorporation: For absolute quantification, include a dilution series of recombinant At5g35200 protein

• Image acquisition parameters: Maintain consistent exposure times and settings across all experimental conditions

• Analysis software: Use dedicated densitometry software with background subtraction capabilities

When publishing results, report both raw and normalized data along with detailed normalization methods to ensure reproducibility and transparency .

What statistical approaches are most appropriate for analyzing At5g35200 expression across developmental stages or stress conditions?

The appropriate statistical analysis depends on experimental design and data characteristics:

• For comparing two conditions (e.g., control vs. treatment):

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-parametric analysis

• For multiple conditions or time points:

  • One-way ANOVA followed by Tukey's post-hoc test for normally distributed data

  • Kruskal-Wallis followed by Dunn's test for non-parametric analysis

• For developmental time-series data:

  • Repeated measures ANOVA with appropriate post-hoc tests

  • Mixed-effects models for incomplete datasets

• For dealing with outliers:

  • Apply Grubbs' test or ROUT method before deciding whether to include or exclude data points

  • Always report all exclusions transparently

• For correlation analysis:

  • Pearson correlation for linear relationships between normally distributed variables

  • Spearman rank correlation for non-linear or non-parametric relationships

Sample size calculation should be performed before experiments to ensure adequate statistical power, typically aiming for 80-90% power with α=0.05 .

How might At5g35200 antibody be adapted for emerging single-cell proteomics applications?

As single-cell proteomics emerges as a frontier technology in plant biology, At5g35200 antibody could be adapted through several innovative approaches:

• Antibody conjugation strategies:

  • Direct labeling with fluorophores or mass cytometry tags for single-cell detection

  • Conjugation to DNA barcodes for single-cell antibody sequencing approaches

  • Attachment to nanoparticles for signal amplification in low-abundance settings

• Microfluidic integration:

  • Incorporation into droplet-based single-cell isolation platforms

  • Application in microfluidic antibody capture arrays

  • Development of continuous-flow antibody detection systems

• Signal amplification technologies:

  • Proximity extension assays for improved sensitivity

  • Immuno-PCR adaptations for digital quantification

  • Tyramide signal amplification for visualization in tissue contexts

These adaptations could transform At5g35200 antibody from a conventional research tool into a component of cutting-edge approaches for understanding protein heterogeneity at cellular resolution in complex plant tissues .

What potential exists for using At5g35200 antibody in conjunction with CRISPR-engineered variants to study protein structure-function relationships?

The combination of At5g35200 antibody with CRISPR-engineered protein variants presents powerful opportunities for structure-function studies:

• Epitope accessibility mapping:

  • Generate a library of CRISPR-edited plants with systematic mutations across the At5g35200 protein

  • Use the antibody to determine which mutations affect epitope recognition

  • Correlate structural changes with functional outcomes

• Domain-specific function analysis:

  • Create truncation or domain-swap variants using CRISPR

  • Use the antibody to confirm expression and localization of engineered proteins

  • Correlate antibody binding patterns with phenotypic outcomes

• Post-translational modification studies:

  • Generate CRISPR-edited plants with mutations at predicted modification sites

  • Use the antibody alongside modification-specific antibodies to assess relationships

  • Develop a comprehensive model of how modifications affect protein function

• Protein-protein interaction interface mapping:

  • Create CRISPR variants with mutations at predicted interaction interfaces

  • Use the antibody in co-immunoprecipitation studies to assess interaction changes

  • Build structural models of protein complexes based on interaction data

These approaches represent the frontier of integrating antibody-based detection with genome editing technologies in plant molecular biology research .