APRR5 Antibody

What is APRR5 and what is its biological function?

APRR5 is a pseudo-response regulator protein involved in plant circadian rhythm regulation. It functions as a transcriptional repressor of clock-controlled genes including CCA1 and LHY. APRR5 mutations affect various circadian-associated biological processes including flowering time in long-day photoperiods, red light sensitivity during early photomorphogenesis, and the period of free-running rhythms of certain clock genes in constant white light. Additionally, APRR5 works additively with Evening Complex (EC), PRR7, and PRR9 to regulate hypocotyl growth under photoperiodic conditions .

What types of APRR5 antibodies are currently available for plant research?

Several types of APRR5 antibodies are currently available for research applications:

• Rabbit polyclonal antibodies to APRR5:

  • Validated specifically for Western Blot applications with Arabidopsis thaliana samples

  • Typically recommended at dilutions of 1:500 to 1:1,000 for optimal results

• Anti-Two-component response regulator-like APRR5 Antibody:

  • Available in lyophilized form for extended shelf life

  • Shows cross-reactivity with multiple species including Arabidopsis thaliana, Brassica napus, and Brassica rapa

  • Different product variants (e.g., PHY3417A and PHY3548A) with slightly different species reactivity profiles

These antibodies are designed primarily for detecting APRR5 in plant research focused on circadian biology and related physiological processes.

How should APRR5 antibodies be properly stored to maintain activity?

For optimal maintenance of APRR5 antibody activity, the following storage protocols are recommended:

• Store lyophilized antibody according to manufacturer's specific instructions

• Use a manual defrost freezer to avoid temperature fluctuations

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Upon receipt, immediately store at the recommended temperature (typically -20°C for long-term storage)

• For working solutions, store small aliquots at recommended temperatures (usually 4°C for short-term use)

For reconstituted antibodies, consider adding stabilizers like BSA (0.1-1%) or glycerol (10-50%) to prevent denaturation during storage and minimize freeze-thaw damage.

What is the optimal Western blotting protocol for detecting APRR5 in plant samples?

Optimized Western Blotting Protocol for APRR5 Detection:

• Sample Preparation:

  • Extract total protein from Arabidopsis tissue using a buffer containing protease inhibitors

  • For nuclear-localized APRR5, consider nuclear extraction protocols

  • Harvest tissue at appropriate circadian time points (APRR5 expression follows circadian patterns)

• Gel Electrophoresis:

  • Load 20-40 μg of total protein per lane on a 10% SDS-PAGE gel

  • Include molecular weight markers (APRR5 is approximately 58-60 kDa)

• Transfer and Blocking:

  • Transfer proteins to PVDF membrane (preferable for higher protein binding capacity)

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Incubate with anti-APRR5 antibody at 1:500 dilution in blocking buffer overnight at 4°C

  • Wash 3-4 times with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5,000 to 1:10,000 dilution for 1 hour

• Detection:

  • Develop using ECL substrate

  • For low abundance detection, use enhanced chemiluminescence systems

  • Expected band size: 58-60 kDa for APRR5, with possible additional bands representing post-translational modifications

How can researchers validate the specificity of APRR5 antibodies?

A comprehensive validation strategy for APRR5 antibodies should include:

• Genetic Controls:

  • Compare antibody reactivity between wild-type plants and aprr5 knockout/knockdown mutants

  • Test in plants overexpressing APRR5 as positive controls

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess synthetic peptide corresponding to the immunogen

  • Compare signal between competed and non-competed antibody applications

  • Specific binding should be significantly reduced in the competed samples

• Cross-Reactivity Assessment:

  • Test antibody against recombinant APRR5 and related PRR family proteins

  • Verify specificity across species: The antibody should show expected reactivity in Arabidopsis thaliana, Brassica napus, and Brassica rapa as documented

• Temporal Expression Patterns:

  • Verify that detected protein follows the expected circadian expression pattern of APRR5

  • Compare protein detection with known mRNA expression data

A systematic validation approach should document all these aspects in a laboratory notebook with detailed experimental conditions and results.

What approaches can be used to detect low-abundance APRR5 protein in plant samples?

For detecting low-abundance APRR5 protein, researchers should consider these methodological improvements:

• Sample Enrichment:

  • Perform subcellular fractionation to isolate nuclear fractions where APRR5 functions

  • Consider immunoprecipitation with anti-APRR5 antibodies to concentrate the protein before analysis

• Signal Amplification Methods:

  • Use high-sensitivity ECL detection systems designed for low-abundance proteins

  • Consider tyramide signal amplification (TSA) for immunohistochemistry applications

  • Employ biotin-streptavidin amplification systems for enhanced detection

• Western Blot Optimization:

  • Increase antibody concentration (1:250 to 1:500 range)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use PVDF membranes instead of nitrocellulose for higher protein binding capacity

  • Consider adding 0.1% SDS to transfer buffer to improve high molecular weight protein transfer

• Timing Considerations:

  • Sample at appropriate circadian time points when APRR5 expression is highest

  • Compare expression across multiple time points to identify optimal sampling windows

These approaches can be combined as needed depending on the specific research context and equipment availability.

How can APRR5 antibodies be used to investigate protein-protein interactions in the plant circadian clock?

APRR5 antibodies can facilitate several advanced protein interaction studies:

• Co-Immunoprecipitation (Co-IP):

  • Use anti-APRR5 antibodies to pull down APRR5 complexes from plant extracts

  • Extract proteins using gentle buffers that preserve protein-protein interactions

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Look for interactions with known clock components like TOC1, PRR7, PRR9, CCA1, and LHY

• Chromatin Immunoprecipitation (ChIP):

  • Since APRR5 functions as a transcriptional repressor, ChIP can identify direct binding targets

  • Cross-link proteins to DNA in planta using formaldehyde

  • Immunoprecipitate with anti-APRR5 antibody

  • Analyze enriched DNA regions by qPCR or sequencing (ChIP-seq)

  • Map APRR5 binding sites on target gene promoters

• Proximity Ligation Assay (PLA):

  • Detect protein-protein interactions with spatial resolution in intact tissues

  • Combine anti-APRR5 antibody with antibodies against potential interaction partners

  • Visualize interactions as discrete fluorescent spots

  • Particularly valuable for detecting interactions that may be lost in extract-based methods

• Temporal Interaction Mapping:

  • Sample tissue at multiple time points across a 24-hour cycle

  • Perform Co-IP with anti-APRR5 antibody at each time point

  • Generate a dynamic interaction network model of clock components

These approaches provide complementary data about APRR5 interactions, with Co-IP being most suitable for stable interactions and PLA better for detecting transient or context-dependent interactions.

What methods can be used to study post-translational modifications of APRR5?

Post-translational modifications of APRR5 can be studied using the following methodological approaches:

• Phosphorylation Analysis:

  • Immunoprecipitate APRR5 using specific antibodies

  • Perform Western blot with anti-APRR5 and anti-phospho-specific antibodies

  • Treat samples with λ-phosphatase to confirm phosphorylation

  • For precise site identification, analyze immunoprecipitated APRR5 by mass spectrometry

• Ubiquitination Detection:

  • Treat plants with proteasome inhibitors (MG132) to accumulate ubiquitinated proteins

  • Immunoprecipitate with anti-APRR5 antibody

  • Probe Western blots with anti-ubiquitin antibodies

  • Look for higher molecular weight bands indicating ubiquitinated forms of APRR5

• Modification Dynamics Analysis:

  • Sample plant tissue at 3-4 hour intervals over a 24-hour cycle

  • Track changes in modification patterns relative to circadian time

  • Create a temporal map of modifications in relation to protein function

  • Correlate modifications with protein stability and interactions

• PTM Site Validation:

  • Generate transgenic plants expressing APRR5 with mutated modification sites

  • Use anti-APRR5 antibodies to confirm expression

  • Analyze phenotypic effects on circadian rhythms and plant development

This multi-faceted approach allows researchers to comprehensively characterize how post-translational modifications regulate APRR5 function within the circadian system.

How can APRR5 antibodies be used in studies of environmental stress responses in plants?

APRR5 antibodies can provide valuable insights into plant stress responses through these approaches:

• Stress-Induced Protein Dynamics:

  • Track APRR5 protein levels during various stresses (heat, cold, drought, etc.)

  • Compare with unstressed controls at the same circadian times

  • Use anti-APRR5 antibodies for Western blot quantification

  • Examine changes in post-translational modification patterns

  • Correlate protein changes with alterations in circadian period/phase

• Stress Memory Studies:

  • Use ChIP with anti-APRR5 antibodies to map binding sites before, during, and after stress

  • Compare binding patterns in primed versus non-primed plants

  • Investigate histone modifications at APRR5-bound promoters

  • Study APRR5's potential role in stress memory formation

• Tissue-Specific Stress Responses:

  • Employ immunohistochemistry with anti-APRR5 antibodies to localize protein in different tissues under stress

  • Compare stress responses in roots versus shoots

  • Examine cell type-specific responses within complex tissues

• Combined Stress Analysis:

  • Examine APRR5 under combined stresses (e.g., heat+drought)

  • Use antibodies to track dynamic responses to stress combinations

  • Compare with single stress responses to identify synergistic or antagonistic effects

These approaches can help uncover how the circadian clock, through components like APRR5, influences stress adaptation mechanisms in plants.

What are common issues encountered when using APRR5 antibodies and how can they be resolved?

Researchers frequently encounter these issues when working with APRR5 antibodies:

When troubleshooting, it's important to systematically test each variable independently while keeping detailed records of changes and outcomes to identify the specific issue.

How should researchers interpret complex banding patterns in APRR5 Western blots?

Complex banding patterns in APRR5 Western blots require careful interpretation:

• Multiple Band Analysis:

  Band Pattern	Likely Interpretation
  Multiple bands near expected MW (58-60 kDa)	Post-translational modifications, particularly phosphorylation
  Higher MW bands (>75 kDa)	Ubiquitinated/SUMOylated forms of APRR5
  Lower MW bands (<50 kDa)	Degradation products or alternative splice variants
  Doublet bands	Often indicate phosphorylation states

• Verification Methods:

  • Treat samples with phosphatase to confirm phosphorylation (bands should collapse to a single band)

  • Use proteasome inhibitors to confirm ubiquitination (higher MW bands should increase)

  • Compare with known knockout/overexpression controls to confirm specificity

  • Perform time-course analysis to determine if bands show circadian patterns

• Temporal Context Consideration:

  • APRR5 expression and modification follow circadian patterns

  • Compare samples collected at the same circadian time point

  • Create a time-course analysis sampling every 4 hours across 24 hours

  • Quantify relative abundance of each band at each time point

Understanding the biological significance of these patterns requires integrating Western blot data with other approaches such as mass spectrometry and genetic studies.

How can researchers quantitatively analyze APRR5 protein levels from Western blot data?

For reliable quantitative analysis of APRR5 from Western blots:

• Image Acquisition:

  • Use a digital imaging system with a linear dynamic range

  • Avoid saturated pixels by optimizing exposure time

  • Capture multiple exposures if necessary

  • Use consistent acquisition settings across comparable experiments

• Normalization Approaches:

  • Select appropriate loading controls:

    • Nuclear proteins for nuclear-localized APRR5

    • Time-stable references (not clock-controlled)

    • Total protein staining (Ponceau S) as an alternative to single protein controls

  • Normalize APRR5 band intensity to loading control

• Analysis Methods:

  • Use integrated density values rather than peak intensity

  • Apply consistent background subtraction

  • Analyze technical triplicates for each biological sample

  • Include at least three biological replicates

  • Apply appropriate statistical tests (ANOVA, t-test)

• Circadian Data Analysis:

  • For time-course data, consider circadian statistics packages (JTK_CYCLE, RAIN)

  • Plot data relative to circadian time or zeitgeber time

  • Calculate rhythm parameters (period, phase, amplitude)

  • Compare with transcript rhythms to identify post-transcriptional regulation

This quantitative approach allows researchers to derive meaningful biological insights from APRR5 protein dynamics data.

How might APRR5 antibodies facilitate studies of plant adaptation to changing climate conditions?

APRR5 antibodies can contribute significantly to climate adaptation research through:

• Temperature Adaptation Studies:

  • Track APRR5 protein responses to temperature extremes and fluctuations

  • Compare thermotolerant versus sensitive varieties using quantitative Western blot analysis

  • Identify beneficial APRR5 alleles or post-translational modifications associated with thermal resilience

  • Study how temperature affects APRR5 protein-protein interactions and DNA binding

• Combined Stress Research:

  • Examine APRR5 under climate-relevant stress combinations (heat+drought, elevated CO₂+heat)

  • Use antibodies to track complex protein dynamics under fluctuating versus constant conditions

  • Investigate how changing day/night temperature differentials affect clock protein function

  • Correlate protein changes with physiological adaptations

• Acclimation Mechanisms:

  • Use ChIP with anti-APRR5 antibodies to map stress-induced changes in genomic binding

  • Investigate APRR5's role in "stress memory" and acclimation processes

  • Compare protein dynamics during repeated versus novel stress exposures

  • Develop models of clock-mediated climate resilience

These approaches can provide mechanistic insights into how plant circadian systems may adapt to or be compromised by changing climate conditions.

What emerging technologies might enhance APRR5 antibody applications in plant research?

Several cutting-edge technologies show promise for advancing APRR5 research:

• Single-Cell Analysis Approaches:

  • Adapting single-cell proteomics methods for plant tissues

  • Using microfluidic platforms for cell-specific APRR5 quantification

  • Correlating with single-cell transcriptomics

  • Examining cell-to-cell variability in clock components

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization of APRR5

  • Expansion microscopy for improved visualization in dense plant tissues

  • Label-free imaging techniques to bypass limitations of antibody penetration

  • Real-time imaging of clock protein dynamics in living plant cells

• Proximity Labeling Technologies:

  • BioID or TurboID fusion proteins to identify transient APRR5 interaction partners

  • APEX2 proximity labeling for mapping spatial protein relationships

  • Integration with antibody validation to confirm interactions

  • Temporal mapping of dynamic protein complexes

• Computational Biology Integration:

  • Machine learning approaches for predicting protein-protein interactions

  • Integrating antibody-derived proteomics with multi-omics datasets

  • Creating predictive models of clock function under environmental change

  • Applying active learning algorithms to optimize experimental design

These technologies can overcome current limitations in studying low-abundance transcription factors like APRR5 in complex plant tissues.

How might APRR5 research contribute to improving crop productivity and resilience?

APRR5 research using antibody-based approaches has significant potential for agricultural applications:

• Optimizing Plant Growth Cycles:

  • Study APRR5's role in photoperiod sensing and flowering time regulation

  • Map protein patterns in high-yielding versus low-yielding cultivars

  • Develop varieties with optimized circadian rhythms for specific geographic regions

  • Engineer improved growth rhythms for yield stability under variable conditions

• Resource-Use Efficiency:

  • Investigate APRR5's involvement in stomatal rhythm regulation using immunolocalization

  • Study protein dynamics during drought using antibody detection methods

  • Compare wild-type versus clock-modified lines for water and nutrient use efficiency

  • Develop crops with improved resource acquisition through optimized timing

• Stress Resilience Enhancement:

  • Characterize APRR5 protein behavior during environmental stresses

  • Identify beneficial variants for breeding stress-tolerant crops

  • Develop molecular markers based on functional protein insights

  • Create circadian-informed protocols for crop management under stress conditions

The circadian clock, including components like APRR5, represents an untapped opportunity for crop improvement by synchronizing plant physiological processes with environmental cycles and stresses.