PER62 Antibody

What is PER62 and why are antibodies against it important in plant research?

PER62 (Peroxidase 62) belongs to the plant peroxidase family that plays crucial roles in cell wall metabolism, defense responses, and stress adaptation . PER62 antibodies are essential tools for studying:

• Root development processes regulated by light perception via the HY5 pathway

• Redox homeostasis in plant stress responses

• Cell wall modifications during development and under stress conditions

PER62, along with related peroxidases (PER6, PER61, PER71, PER73), is known to be repressed in the hy5 mutant and appears to be involved in root apical meristem (RAM) development . Antibodies against PER62 enable researchers to investigate protein localization, expression levels, and post-translational modifications in various experimental contexts.

Which applications are most suitable for PER62 antibodies in plant research?

Based on comparative analysis of antibody applications, PER62 antibodies are most commonly used in the following techniques:

For Western blot applications, researchers should be aware that PER62 typically appears at approximately 35-38 kDa under reducing conditions, though post-translational modifications may alter migration patterns.

How should I design controls for experiments using PER62 antibodies?

Robust experimental design with appropriate controls is crucial for PER62 antibody applications:

• Positive controls:

  • Use tissue/cells known to express PER62 (e.g., root tissues exposed to stress conditions)

  • Recombinant PER62 protein (when available)

  • Transfected cell lines overexpressing PER62

• Negative controls:

  • PER62 knockout/knockdown plant lines

  • Pre-immune serum at equivalent concentrations

  • Primary antibody omission

  • Competitive blocking with immunizing peptide

• Specificity controls:

  • Cross-reactivity assessment with related peroxidases (especially PER61, which shares high sequence homology)

  • Western blot analysis showing single band at expected molecular weight

  • Immunoprecipitation followed by mass spectrometry validation

For genetic studies, comparing wild-type and per62 mutant plants under both control and stress conditions provides the most definitive validation of antibody specificity .

What are the key considerations for optimizing PER62 antibody-based Western blot protocols?

Optimizing Western blot protocols for PER62 detection requires attention to several parameters:

• Sample preparation:

  • Fresh extraction in buffer containing protease inhibitors

  • Addition of reducing agents (e.g., DTT or β-mercaptoethanol)

  • Denaturation temperature: 95°C for 5 minutes optimal

  • Avoid freeze-thaw cycles of protein extracts

• Gel and transfer conditions:

  • 10-12% SDS-PAGE gels provide optimal resolution

  • PVDF membranes generally offer better results than nitrocellulose

  • Semi-dry transfer: 25V for 30 minutes or wet transfer: 100V for 1 hour

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:1000 in 5% BSA or milk in TBST

  • Overnight incubation at 4°C yields best signal-to-noise ratio

  • Secondary antibody: anti-rabbit HRP at 1:5000-1:20000 dilution

• Detection optimization:

  • Enhanced chemiluminescence (ECL) substrates work well for standard applications

  • Fluorescent secondary antibodies provide better quantification options

  • Signal enhancers may be needed for low abundance samples

How can I use PER62 antibodies to study plant stress responses and ROS signaling pathways?

PER62 antibodies can be powerful tools for investigating plant stress responses and reactive oxygen species (ROS) signaling networks:

• Subcellular localization studies:

  • Immunofluorescence with organelle markers to determine changes in PER62 localization during stress

  • Subcellular fractionation followed by Western blot to quantify translocation between compartments

• Stress-responsive post-translational modifications:

  • Immunoprecipitation with PER62 antibodies followed by mass spectrometry

  • Phospho-specific antibodies to detect activation status

  • Analysis of redox-dependent modifications

• Protein-protein interactions:

  • Co-immunoprecipitation to identify PER62 interaction partners

  • Proximity ligation assays to confirm in situ interactions

  • Chromatin immunoprecipitation if PER62 associates with DNA-binding proteins

Research has shown that peroxidases like PER62 can function as either ROS consumers or producers depending on environmental conditions and cellular context . Studies indicate these dual roles are influenced by specific amino acid differences in conserved regions called the "Alpha and Beta Buttons," which affect substrate binding and reaction kinetics.

What approaches should I use to investigate PER62's role in root development and light signaling?

To investigate PER62's function in root development and light signaling:

• Expression pattern analysis:

  • Immunohistochemistry of root sections across developmental stages

  • Compare expression in light vs. dark conditions

  • Analysis of expression in HY5 pathway mutants

• Functional studies:

  • Compare phenotypes between wild-type and per62 mutant roots

  • Analyze PER62 protein levels in different light conditions using Western blot

  • Examine PER62 interaction with HY5 or downstream components

• ROS homeostasis assessment:

  • Combine PER62 immunolocalization with ROS-specific dyes

  • Quantify H₂O₂ and O₂⁻ levels in wild-type vs. per62 mutants

Recent studies have established that PER62, like PER6, is regulated by the transcription factor HY5, which accumulates in light conditions to direct root development . The root-specific light perception pathway demonstrates that roots can perceive light independently from aboveground organs, with HY5 directly binding to promoters of peroxidase genes to control ROS homeostasis.

How can I address non-specific binding issues with PER62 antibodies?

Non-specific binding is a common challenge with plant peroxidase antibodies due to the high homology between family members. To minimize this issue:

• Antibody selection and validation:

  • Choose antibodies raised against unique peptide regions of PER62

  • Validate antibody specificity using recombinant proteins or knockout lines

  • Consider using monoclonal antibodies for higher specificity

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extended blocking times (2-4 hours at room temperature)

  • Addition of 0.1-0.5% Tween-20 or Triton X-100 to reduce hydrophobic interactions

• Advanced approaches for challenging samples:

  • Pre-absorption of antibody with recombinant related peroxidases

  • Dual-color Western blot with antibodies to related peroxidases to identify specific bands

  • Sequential immunoprecipitation to deplete cross-reactive proteins

When analyzing PER62 in complex plant tissues, consider using gradient gels (4-20%) to better resolve proteins of similar molecular weights, as several plant peroxidases migrate similarly on standard gels.

What strategies can improve detection sensitivity for low-abundance PER62 protein?

For low-abundance PER62 detection, implement these sensitivity-enhancing approaches:

• Sample enrichment:

  • Subcellular fractionation to concentrate compartment-specific pools

  • Immunoprecipitation before Western blotting

  • Protein concentration methods (TCA precipitation, acetone precipitation)

• Signal amplification techniques:

  • Tyramide signal amplification for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence substrates with extended exposure

  • Biotin-streptavidin detection systems

• Instrument-based enhancements:

  • Cooled CCD cameras for longer exposures without increased background

  • Laser-based scanning for fluorescent detection

  • Digital stacking of multiple short exposures

• Protocol optimizations:

  • Extended primary antibody incubation (overnight at 4°C or 48 hours)

  • Reduced washing stringency (lower salt concentration)

  • Use of signal enhancers like sodium azide to prolong HRP activity

How should I quantify and normalize PER62 protein levels across different experimental conditions?

Proper quantification and normalization are essential for comparative analysis of PER62 expression:

• Quantification methods:

  • Densitometry analysis of Western blot bands using ImageJ or similar software

  • Fluorescence intensity measurements for immunofluorescence

  • ELISA standard curves for absolute quantification

• Normalization approaches:

  • Housekeeping proteins (tubulin, actin, GAPDH) for Western blots

  • Total protein normalization using stain-free gels or Ponceau staining

  • Ratiometric analysis against internal controls

• Statistical analysis recommendations:

  • Minimum of 3-4 biological replicates

  • Appropriate statistical tests based on data distribution (t-test, ANOVA)

  • Consider logarithmic transformation for data with wide dynamic range

For accurate comparisons between tissue types or treatments, a combination of normalization methods is recommended, as housekeeping protein levels may vary under stress conditions that alter PER62 expression.

How do I interpret conflicting results between transcript and protein levels for PER62?

Discrepancies between PER62 mRNA and protein levels are common and can provide valuable insights:

• Potential mechanisms explaining discrepancies:

  • Post-transcriptional regulation (miRNA targeting, mRNA stability)

  • Translational efficiency differences

  • Post-translational modifications affecting antibody recognition

  • Protein turnover rate variations

• Integrated analysis approach:

  • Temporal analysis combining RT-qPCR and Western blotting across time points

  • Polysome profiling to assess translational efficiency

  • Proteasome inhibitor treatments to evaluate protein stability

  • Analysis of relevant miRNAs targeting PER62 transcripts

• Validation strategies:

  • Reporter constructs with PER62 5' and 3' UTRs to assess post-transcriptional regulation

  • Pulse-chase experiments to determine protein half-life

  • Analysis in mutants of suspected regulatory pathways

Research on plant peroxidases has shown that transcript levels often do not correlate with protein abundance or enzyme activity due to complex regulation at multiple levels . Specifically, stress conditions can trigger rapid changes in peroxidase activity through post-translational modifications without immediate changes in protein levels.

How can I use PER62 antibodies in multiplexed imaging and high-throughput screening applications?

Emerging applications for PER62 antibodies in advanced research settings include:

• Multiplexed immunofluorescence approaches:

  • Sequential immunostaining with antibody stripping between rounds

  • Spectral unmixing of fluorophores with overlapping emission spectra

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

• High-content screening applications:

  • Automated image acquisition and analysis platforms

  • Cell painting assays with PER62 as one of multiple markers

  • Machine learning-based classification of phenotypes

• Single-cell analysis techniques:

  • Flow cytometry of protoplasts labeled with PER62 antibodies

  • Imaging mass cytometry for spatial resolution

  • In situ PLA (proximity ligation assay) to detect specific interactions

These advanced applications require highly specific antibodies with validated performance characteristics and often benefit from recombinant antibody formats with consistent batch-to-batch performance.

What are the considerations for using PER62 antibodies in cross-species investigations?

Cross-species applications require careful consideration of sequence conservation:

• Sequence alignment analysis:

  • Multiple sequence alignment of PER62 orthologs across species

  • Identification of conserved epitopes recognized by the antibody

  • Phylogenetic analysis to predict cross-reactivity

• Validation strategies for cross-species use:

  • Western blot analysis of recombinant proteins from target species

  • Testing in species with known sequence divergence

  • Epitope mapping to confirm antibody binding sites

• Optimization for divergent species:

  • Adjustment of antibody concentration

  • Modified blocking conditions

  • Species-specific secondary antibodies

The peroxidase family is well-conserved across plant species, but specific epitopes may vary. When using PER62 antibodies across species, researchers should focus on antibodies targeting the most conserved regions, particularly around the catalytic site which is highly preserved across the peroxidase family.