PER31 Antibody

What is PER31 and what systems express this protein?

PER31 (Peroxidase 31) is a plant protein belonging to the peroxidase superfamily, primarily studied in Arabidopsis thaliana (Mouse-ear cress). It functions as an enzyme with peroxidase activity (EC 1.11.1.7) and is involved in oxidation-reduction processes within plant tissues. The protein is encoded by the AT3G28200 gene and is also known by alternative names including Atperox P31 and ATP41 . Unlike the similarly named PER3 (Period Circadian Clock 3) in humans, plant PER31 serves entirely different functions related to oxidative stress responses and cell wall modifications rather than circadian rhythm regulation.

What applications are PER31 antibodies validated for?

Based on the available research materials, PER31 antibodies have been validated for several key applications in plant research. These primarily include: Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection, and Western Blot (WB) for protein identification and semi-quantitative analysis . The validation typically involves confirming specific binding to recombinant Arabidopsis thaliana PER31 protein and verifying minimal cross-reactivity with other peroxidase family members. While these are the primary validated applications, researchers should be aware that optimization may be required for additional techniques such as immunohistochemistry in plant tissues.

What is the difference between PER31 antibody and PER3 antibody?

Although their names are similar, these antibodies target completely different proteins in different organisms:

Researchers must be careful not to confuse these distinct antibodies when designing experiments, as they target entirely different biological systems .

How should I optimize Western blot protocols for PER31 antibody detection?

For optimal Western blot results with PER31 antibody in plant research:

• Sample preparation: Extract proteins from Arabidopsis tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. Homogenize tissues thoroughly on ice.

• Protein separation: Load 20-50μg of total protein per lane on a 10-12% SDS-PAGE gel. Include positive controls (recombinant PER31) and negative controls.

• Transfer and blocking: After electrophoresis, transfer proteins to a PVDF membrane and block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute PER31 antibody at 1:500-1:5000 in blocking buffer and incubate overnight at 4°C. The optimal dilution should be determined empirically for each antibody lot .

• Washing and detection: Wash the membrane with TBST (3-5 times, 5 minutes each) and incubate with an appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution. After washing, visualize using enhanced chemiluminescence.

• Expected results: PER31 typically appears as a band around 35-40 kDa. Verify specificity using a pre-immune serum control or competitive blocking with the immunizing peptide.

What steps should be taken to validate PER31 antibody specificity in plant research?

Comprehensive validation of PER31 antibody specificity is critical for generating reliable research data:

• Recombinant protein validation: Test antibody against purified recombinant PER31 protein to confirm binding to the target antigen .

• Knockout/knockdown controls: When available, use per31 mutant or knockdown Arabidopsis lines as negative controls to confirm antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to samples. Specific signals should be significantly reduced or eliminated.

• Cross-reactivity assessment: Test the antibody against related peroxidase family members to ensure minimal cross-reactivity.

• Multiple detection methods: Confirm findings using at least two independent detection methods (e.g., Western blot and immunohistochemistry).

• Antibody lot validation: Each new lot should be validated against previous lots to ensure consistent specificity and sensitivity.

• Signal-to-noise ratio optimization: Titrate antibody concentrations to determine the optimal dilution that maximizes specific signal while minimizing background.

How can PER31 antibody be utilized in studying plant stress responses?

PER31 antibody can be leveraged as a powerful tool for investigating plant oxidative stress responses through several approaches:

• Expression profiling: Use Western blot analysis with PER31 antibody to quantify changes in protein expression across different stress conditions (drought, salinity, pathogen exposure). This allows tracking of peroxidase induction as part of the plant's defense mechanism.

• Tissue localization studies: Employ immunohistochemistry or immunofluorescence with optimized PER31 antibody protocols to visualize the spatial distribution of PER31 in different plant tissues under stress conditions.

• Enzymatic activity correlation: Combine antibody-based detection of PER31 protein levels with peroxidase activity assays to establish relationships between protein abundance and functional activity.

• Protein complex identification: Utilize the antibody for immunoprecipitation (IP) followed by mass spectrometry to identify stress-induced protein interaction partners of PER31.

• Post-translational modification analysis: Use PER31 antibodies in conjunction with modification-specific detection methods to investigate how oxidative stress affects post-translational modifications of the protein.

• Comparative analysis across species: Examine cross-reactivity with homologous proteins in other plant species to study evolutionary conservation of stress response mechanisms.

This multifaceted approach allows researchers to build a comprehensive understanding of how PER31 functions within the broader context of plant stress physiology .

What considerations should be made when designing immunoprecipitation experiments with PER31 antibody?

Successful immunoprecipitation (IP) with PER31 antibody requires careful attention to several critical parameters:

• Antibody quality assessment: Verify that your PER31 antibody is suitable for IP applications, as not all antibodies that work in Western blots will perform well in IP. Antigen-affinity purified antibodies typically provide better results .

• Cross-linking considerations: For studying transient interactions, consider using reversible cross-linking reagents like DSP (dithiobis(succinimidyl propionate)) to stabilize protein complexes before cell lysis.

• Extraction buffer optimization:

  • For membrane-associated complexes: Use buffers containing 1% NP-40 or Triton X-100

  • For nuclear complexes: Include DNase I treatment

  • Always include protease and phosphatase inhibitors

• Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Controls to include:

  • IgG isotype control to identify non-specific binding

  • Input control (5-10% of lysate) to confirm protein presence

  • When available, PER31 knockout/knockdown samples as negative controls

• Washing stringency balance: Use sufficient washing steps to reduce background while preserving specific interactions. A common approach is to perform 3-4 washes with decreasing salt concentrations.

• Elution method selection: Choose between native elution (with competing peptide) or denaturing elution (with SDS buffer), depending on downstream applications.

• Verification strategy: Confirm successful IP by Western blot using a different PER31 antibody recognizing a separate epitope when possible.

How should researchers address inconsistent results when using PER31 antibody across different experimental systems?

Inconsistent results with PER31 antibody can stem from multiple sources. Here's a systematic approach to troubleshooting:

• Antibody storage and handling:

  • Verify proper storage at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles (aliquot upon first use)

  • Check for precipitates before use

  • Note the specific buffer composition (contains 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300)

• Protocol standardization:

  • Document exact conditions for each experiment

  • Standardize protein extraction methods

  • Use consistent blocking agents and incubation times

  • Maintain identical washing procedures

• Sample variability assessment:

  • Growth conditions of plants may affect PER31 expression

  • Developmental stage impacts peroxidase expression patterns

  • Tissue-specific expression differences require standardized sampling

• Technical optimization:

  • Titrate antibody concentration (typically 1:500-1:5000 for WB)

  • Adjust exposure times for consistent signal-to-noise ratio

  • Consider enhanced antigen retrieval methods for fixed tissues

• Antibody lot variation:

  • Compare lot numbers when results differ

  • Maintain reference samples to test new antibody lots

  • Request specification sheets from manufacturers

• Cross-validation approach:

  • Verify key findings with alternative antibodies when available

  • Complement antibody-based results with mRNA expression data

  • Consider orthogonal detection methods

• Data normalization:

  • Use consistent loading controls

  • Apply quantitative analysis software

  • Present data as relative changes rather than absolute values

What are the potential causes and solutions for high background when using PER31 antibody in immunohistochemistry?

High background is a common challenge when adapting PER31 antibodies for immunohistochemistry in plant tissues. Here's a systematic approach to diagnose and resolve this issue:

Implementing a systematic approach by changing one variable at a time and maintaining detailed records of optimization steps will help determine the most effective protocol for your specific experimental conditions .

How does the performance of PER31 antibody compare with other methods for studying plant peroxidases?

When evaluating different techniques for studying PER31 and other plant peroxidases, researchers should consider the relative advantages and limitations of each approach:

• Antibody-based detection vs. Activity assays:

  • Antibody detection (Western blot, ELISA) specifically quantifies PER31 protein levels regardless of enzymatic activity

  • Peroxidase activity assays (using substrates like DAB or guaiacol) measure total peroxidase function but cannot distinguish individual isoforms

  • Combined approaches provide insights into the relationship between protein abundance and functional activity

• Immunological detection vs. Transcriptional analysis:

  • PER31 antibody detection reveals post-transcriptional regulation and protein stability factors

  • RT-qPCR for PER31 mRNA provides insights into transcriptional regulation

  • RNA-seq offers broader context but with less specific quantification

  • Discrepancies between protein and mRNA levels can reveal important regulatory mechanisms

• Traditional immunoblotting vs. Multiplex protein analysis:

  • Standard Western blot with PER31 antibody offers reliable single-protein quantification

  • Multiplex immunoassays allow simultaneous detection of multiple peroxidases

  • Mass spectrometry provides comprehensive proteomic profiling but may miss low-abundance isoforms

• Antibody applications vs. CRISPR-based approaches:

  • PER31 antibody detection works with wild-type plants without genetic modification

  • CRISPR-mediated tagging (e.g., FLAG, GFP) allows live imaging but may affect protein function

  • Knockout/knockdown lines provide loss-of-function context but may have compensatory mechanisms

• Species-specific considerations:

  • PER31 antibodies raised against Arabidopsis may have variable cross-reactivity with other plant species

  • Evolutionary divergence of peroxidases requires careful validation across species

Each approach provides complementary information, and combining multiple techniques often yields the most comprehensive understanding of PER31 biology in plant research .

What emerging techniques might enhance the utility of PER31 antibody in plant research?

Several cutting-edge methodologies are expanding the research applications of plant antibodies like anti-PER31:

• Proximity labeling techniques:

  • BioID or TurboID fusion with PER31 combined with antibody pulldown can map protein interaction networks

  • APEX2-based proximity labeling can identify transient interaction partners in native cellular contexts

  • These approaches reveal the broader functional complexes in which PER31 operates

• Super-resolution microscopy:

  • STORM or PALM microscopy using fluorescently-labeled PER31 antibodies can visualize subcellular localization at nanometer resolution

  • Expansion microscopy physically enlarges samples for enhanced resolution with standard confocal microscopy

  • These techniques reveal previously undetectable spatial organization of PER31 in plant cell walls and membranes

• Single-cell antibody-based detection:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies enables high-dimensional analysis at single-cell resolution

  • Microfluidic platforms for single-cell Western blotting can detect PER31 expression heterogeneity

  • These approaches capture cell-to-cell variability in PER31 expression within tissues

• Quantitative multiplex immunofluorescence:

  • Cyclic immunofluorescence (CycIF) permits sequential staining with multiple antibodies

  • Multiplexed ion beam imaging (MIBI) allows simultaneous detection of dozens of proteins

  • These methods contextualize PER31 expression within broader signaling networks

• Native complex analysis:

  • Blue native PAGE combined with PER31 antibody detection preserves and identifies native protein complexes

  • Cross-linking mass spectrometry (XL-MS) after immunoprecipitation maps precise interaction interfaces

  • These techniques maintain physiologically relevant protein associations

• In situ proximity ligation assay (PLA):

  • Detects protein-protein interactions in fixed samples using paired antibodies

  • Can reveal cell-specific interaction patterns of PER31 with partner proteins

  • Provides spatial context for protein interactions within intact tissues

Researchers can significantly enhance the information gained from PER31 antibodies by adapting these emerging technologies to plant systems, potentially revealing new aspects of peroxidase biology in stress responses and development.