At2g16220 Antibody

What is the At2g16220 protein in Arabidopsis thaliana, and why is studying it important?

At2g16220 encodes a protein in Arabidopsis thaliana that has been implicated in stress response pathways. The protein (UniProt: Q8S8C7) is of interest to plant biologists studying osmotic stress responses, similar to experimental designs used in hyperosmotic priming studies . Research approaches typically involve:

• Characterizing protein expression patterns across different tissues

• Examining regulation under various stress conditions

• Analyzing protein-protein interactions to determine functional pathways

Methodologically, studying At2g16220 requires validated antibodies for detecting native protein expression patterns, essential for correlating transcriptional data with actual protein abundance.

What are the key characteristics of commercially available At2g16220 antibodies?

Commercial At2g16220 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana At2g16220 protein . Key characteristics include:

Methodologically, researchers should note that polyclonal antibodies contain a mixture of antibodies recognizing different epitopes on the target protein, providing robust detection but potentially introducing variability between antibody lots.

How should At2g16220 antibody be stored and handled for optimal performance?

For optimal performance of At2g16220 antibodies:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation and loss of antibody function

• For working solutions, store at 4°C for short-term use (1-2 weeks)

• Prepare aliquots for single use to minimize freeze-thaw cycles

• When preparing dilutions, use buffers containing carrier proteins (e.g., 1% BSA) to prevent adhesion to tube walls

When evaluating antibody performance issues, storage conditions should be the first variable examined, as improperly stored antibodies can result in false negatives or increased background.

What controls should be included when validating At2g16220 antibody specificity?

When validating At2g16220 antibody specificity, the following controls are essential based on established antibody validation methods :

Positive controls:

• Recombinant At2g16220 protein

• Arabidopsis tissues or cells known to express At2g16220

• Arabidopsis plants with At2g16220 overexpression

Negative controls:

• At2g16220 knockout/knockdown lines

• Pre-immune serum (matching the host animal)

• Blocking peptide competition assay

• Non-expressing tissues or developmental stages

Cross-reactivity assessment:

• Testing against closely related proteins in Arabidopsis

• Testing against homologs in related plant species

• Cross-adsorption experiments with purified proteins

What is the recommended protocol for Western blot analysis using At2g16220 antibody?

Based on standard protocols for plant protein analysis, the following methodological approach is recommended:

Sample preparation:

• Extract total protein from Arabidopsis tissues using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Clarify lysates by centrifugation (14,000 × g, 15 min, 4°C)

• Quantify protein concentrations (Bradford or BCA assay)

SDS-PAGE and transfer:

• Load 20-50 μg protein per lane

• Separate proteins on 10-12% SDS-PAGE

• Transfer to PVDF or nitrocellulose membrane (100V, 1 hour)

Immunoblotting:

• Block membrane with 5% non-fat milk in TBST (1 hour, room temperature)

• Incubate with At2g16220 antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST (10 min each)

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

• Wash 3× with TBST (10 min each)

• Develop using ECL reagent

This methodological approach should be optimized based on protein expression levels and specific antibody characteristics.

How can At2g16220 antibody be used for immunohistochemistry in plant tissues?

For immunohistochemistry in plant tissues:

Tissue preparation:

• Fix fresh tissues in 4% paraformaldehyde in PBS (pH 7.4) for 4 hours

• Dehydrate through an ethanol series (30%, 50%, 70%, 90%, 100%)

• Clear in xylene and embed in paraffin

• Section at 5-8 μm thickness

Immunohistochemistry procedure:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval (10 mM sodium citrate, pH 6.0, microwave 10 min)

• Block endogenous peroxidase (3% H₂O₂, 10 min)

• Block with 5% normal goat serum in PBS (1 hour)

• Incubate with At2g16220 antibody (1:100-1:500) overnight at 4°C

• Wash 3× with PBS

• Incubate with biotinylated secondary antibody (1 hour)

• Apply streptavidin-HRP complex

• Develop with DAB substrate

• Counterstain, dehydrate, and mount

When analyzing results, include positive and negative controls in adjacent sections to confirm signal specificity.

How can At2g16220 antibody be used in ChIP-seq experiments to study protein-DNA interactions?

For Chromatin Immunoprecipitation sequencing (ChIP-seq) applications:

ChIP-seq protocol for plant tissues:

• Cross-link proteins to DNA (1% formaldehyde, 10 min)

• Quench with 0.125 M glycine (5 min)

• Extract nuclei and sonicate chromatin (200-500 bp fragments)

• Pre-clear with protein A/G beads

• Immunoprecipitate with At2g16220 antibody (4-10 μg per reaction)

• Wash stringently to remove non-specific interactions

• Reverse cross-links and purify DNA

• Prepare libraries for next-generation sequencing

Based on research with other plant proteins , epitope accessibility in chromatin complexes may be limited. Successful ChIP-seq experiments with At2g16220 antibody would require:

• Confirmation of antibody specificity in nuclear extracts

• Optimization of sonication conditions for Arabidopsis tissues

• Validation of enrichment by qPCR of known targets before sequencing

• Inclusion of appropriate controls (input chromatin, IgG control, etc.)

Bioinformatic analysis should account for the plant genome structure and modification patterns unique to Arabidopsis.

How can At2g16220 antibody be used to study protein-protein interactions in stress response pathways?

For studying protein-protein interactions:

Co-immunoprecipitation (Co-IP) approach:

• Extract proteins under native conditions using:

  • 50 mM HEPES (pH 7.5)

  • 150 mM NaCl

  • 0.5% NP-40

  • 1 mM EDTA

  • Protease/phosphatase inhibitors

• Pre-clear lysate with protein A/G beads

• Immunoprecipitate with At2g16220 antibody (overnight, 4°C)

• Wash beads extensively

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

Proximity-dependent labeling approaches:

• Express At2g16220 fused to BioID or TurboID in Arabidopsis

• After biotin treatment, use At2g16220 antibody to confirm proper expression/localization

• Purify biotinylated proteins and identify by mass spectrometry

When analyzing stress response pathways, differential interactome analysis under normal and stress conditions (e.g., osmotic stress, salt stress) can reveal context-dependent interactions similar to those observed in hyperosmotic priming studies .

What methodological considerations are important when using At2g16220 antibody for quantitative proteomics?

For quantitative proteomics applications:

Sample preparation:

• Extract total protein from control and experimental tissues

• Verify At2g16220 detection by Western blot

• Perform immunoprecipitation with At2g16220 antibody

• Process samples for MS analysis (tryptic digestion, etc.)

Quantification approaches:

• Label-free quantification

• Tandem mass tag (TMT) labeling

• SILAC labeling (for cell culture)

Data analysis considerations:

• Multiple biological replicates (n≥3) are essential

• Include appropriate statistical methods for differential expression analysis

• Consider both fold change and statistical significance (p-value)

• Validate key findings using orthogonal methods (Western blot, qPCR)

When interpreting proteomic data, consider that antibody-based enrichment may introduce biases. Cross-validation with total proteome analysis can help mitigate this limitation.

What are common issues with At2g16220 antibody specificity and how can they be addressed?

Common specificity issues with plant antibodies include :

The research literature indicates that commercially available antibodies for plant proteins can be highly variable in specificity . Crucial validation steps include:

• Testing the antibody in tissues from knockout/knockdown plants

• Performing peptide competition assays

• Confirming that the detected protein size matches the predicted molecular weight

How should researchers interpret differences in At2g16220 protein levels across different experimental conditions?

When interpreting At2g16220 protein level differences:

Quantification approaches:

• Use appropriate normalization controls (loading controls like actin or GAPDH)

• Employ software for densitometric analysis (ImageJ, etc.)

• Present data as fold change relative to control conditions

Statistical analysis:

• Perform experiments with ≥3 biological replicates

• Use appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

• Report p-values and variance measures (standard deviation or standard error)

Biological interpretation:

• Correlate protein levels with transcriptional data (qPCR, RNA-seq)

• Consider post-transcriptional regulation mechanisms

• Examine protein stability under different conditions

When publishing results, follow the guidelines for presenting protein expression data in scientific papers , including properly formatted tables with appropriate statistical measures.

How can researchers distinguish between specific and non-specific binding in antibody-based experiments?

To distinguish specific from non-specific binding:

Experimental approaches:

• Blocking peptide competition: Pre-incubate antibody with excess antigen peptide before application

• Knockout/knockdown validation: Test in tissues lacking the target protein

• Cross-adsorption: Pre-adsorb antibody with related proteins

• Concentration gradient: Test multiple antibody dilutions to optimize signal-to-noise ratio

• Alternative antibodies: Compare results using antibodies targeting different epitopes

Research has shown that cross-blocking experiments can reveal antibody specificity patterns . In these experiments:

• Different antibody clones are used competitively

• Binding patterns reveal epitope relationships

• Non-competitive binding indicates recognition of different epitopes

For data presentation, include both positive and negative controls alongside experimental samples to demonstrate specificity within the actual experimental context.

How might At2g16220 antibody be used in multiplexed proteomic analyses?

For multiplexed proteomic analyses:

Multiplexed immunofluorescence approaches:

• Conjugate At2g16220 antibody to a specific fluorophore or use secondary antibody labeling

• Combine with antibodies against other proteins of interest (using compatible species/isotypes)

• Apply tyramide signal amplification for low-abundance proteins

• Image using confocal microscopy with appropriate controls for spectral overlap

Mass cytometry (CyTOF) applications:

• Conjugate At2g16220 antibody to metal isotopes

• Combine with other metal-tagged antibodies

• Analyze single-cell protein expression patterns

Proximity ligation assays:

• Combine At2g16220 antibody with antibodies against potential interacting partners

• Detect protein-protein interactions in situ with subcellular resolution

These approaches could be particularly valuable for understanding At2g16220 protein function in the context of stress response networks similar to those described in hyperosmotic priming studies .

What considerations are important when using At2g16220 antibody in combination with other techniques like CRISPR gene editing?

When combining antibody detection with CRISPR-edited Arabidopsis:

Key considerations:

• Epitope preservation: Ensure CRISPR edits don't alter the epitope recognized by the antibody

• Validation in edited lines: Confirm antibody specificity in edited backgrounds

• Tagged protein detection: For knock-in tags (FLAG, HA, etc.), compare native protein detection with tag detection

• Mosaic tissues: Account for cellular heterogeneity in partially edited tissues

Experimental workflow:

• Validate CRISPR edits by sequencing

• Confirm protein modification/knockout by Western blot with At2g16220 antibody

• Use antibody to assess protein localization in edited backgrounds

• Compare phenotypes with protein expression patterns

For domain-specific CRISPR edits, consider using multiple antibodies targeting different epitopes to comprehensively assess protein modification.

How can researchers integrate data from At2g16220 antibody-based studies with other 'omics approaches?

For multi-omics integration:

Methodological approaches:

• Correlative analysis: Compare protein levels (detected by At2g16220 antibody) with:

  • Transcriptomic data (RNA-seq)

  • Epigenomic profiles (ChIP-seq for histone modifications)

  • Metabolomic changes related to stress responses

• Network analysis: Place At2g16220 protein in functional networks using:

  • Protein-protein interaction data from immunoprecipitation studies

  • Genetic interaction data from mutant studies

  • Pathway enrichment analysis methods like WGCNA (weighted gene co-expression network analysis)

• Temporal studies: Track protein dynamics during stress responses:

  • Compare with transcriptional kinetics

  • Assess post-translational modifications

  • Correlate with physiological responses

When presenting integrated data, use visualization methods that clearly show relationships between different data types, such as heatmaps for expression data combined with protein interaction networks.