PRR5 Antibody

What is PRR5 and why is it significant in research?

PRR5 exists in two distinct contexts that have important implications for experimental design:

In mammals: PRR5 (Proline-rich protein 5, also known as Protor-1) is a 388 amino acid protein that functions as a component of mTORC2 (mammalian target of rapamycin complex 2). It is widely expressed and contains two RICTOR interaction sites along with a C-terminal Proline-rich region. PRR5 promotes mTORC2 activity and plays a role in regulating cell growth and survival pathways . Human PRR5 has multiple splice variants, with observed molecular weights ranging from 42-50 kDa .

In plants: PRR5 (PSEUDO-RESPONSE REGULATOR 5) acts as a transcriptional repressor in the circadian clock system of Arabidopsis thaliana. It directly regulates gene expression by binding to upstream regions of target genes through its CCT domain . The plant PRR5 protein contains a Pseudo-Receiver (PR) domain that interacts with the F-box protein ZEITLUPE (ZTL), which mediates its ubiquitination and degradation .

Understanding which PRR5 ortholog you're studying is critical for experimental design and antibody selection.

For optimal performance and longevity of PRR5 antibodies:

• Store at -20°C for long-term storage (stable for one year after shipment)

• Short-term storage at 4°C is possible for up to three months

• Avoid repeated freeze-thaw cycles as this can damage antibody integrity

• Most PRR5 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations may contain BSA as a stabilizer (e.g., 0.1% BSA in smaller aliquots)

• Aliquoting is often unnecessary for -20°C storage but recommended if repeated use is anticipated

Improper storage can lead to decreased antibody performance and inconsistent experimental results.

How can I validate the specificity of a PRR5 antibody?

Thorough validation is essential for reliable PRR5 detection. Implement these methodological approaches:

• Genetic validation: Use PRR5 knockout/knockdown systems:

  • Compare antibody signals between wild-type and PRR5-deficient samples (e.g., PRR5 siRNA-treated cells)

  • For plant research, utilize prr5-1 mutant lines as negative controls

• Molecular weight verification:

  • Human PRR5 should appear at approximately 42-50 kDa (primary band)

  • Be aware that observed molecular weights (33 kDa, 50 kDa) may differ from calculated weights (31 kDa, 43 kDa) due to post-translational modifications

• Peptide competition assay:

  • Pre-incubate the antibody with its immunizing peptide before application

  • Signal should be significantly reduced or eliminated in the presence of the blocking peptide

• Expression system validation:

  • Test antibody against recombinant PRR5 or PRR5-tagged constructs (e.g., PRR5-FLAG)

  • Compare endogenous signal with overexpressed PRR5

• Cross-reactivity assessment:

  • Evaluate potential cross-reactivity with other Protor family members

  • Verify expected tissue expression patterns (PRR5 is widely expressed but may show tissue-specific variations)

What are the key considerations when studying PRR5 protein-protein interactions?

When investigating PRR5 interactions, consider these methodological approaches based on the experimental context:

For mammalian PRR5 (mTORC2 component):

• PRR5 forms part of mTORC2 through interaction with RICTOR and SIN1

• When designing co-immunoprecipitation experiments, use mild lysis conditions to preserve protein complexes

• For mapping interaction domains, consider that PRR5 contains two RICTOR-interaction sites (aa 10-95 and 188-218)

• GST-PRR5L pull-down assays have successfully demonstrated binding to mTOR via SIN1 and/or Rictor

For plant PRR5 (circadian regulator):

• The Pseudo-Receiver (PR) domain interacts directly with ZTL for protein degradation

• The CCT domain is essential for DNA binding and transcriptional regulation

• For in vitro validation, fusion proteins such as MBP-PRR5 have been used successfully in pull-down assays

• When studying DNA-protein interactions, consider ChIP approaches targeting the CCT motif

Domain-specific insights for experimental design:

• For studying interaction with ZTL, focus on the PR domain of PRR5

• For analyzing DNA binding properties, focus on the CCT domain

• In plants, PRR5 interacts with ABI5 through its C-terminal fragment, while the bZIP domain of ABI5 is required for this interaction

How do I interpret conflicting data about PRR5 function?

Conflicting data regarding PRR5 function may arise from:

• Context-dependent effects: PRR5 functions differently in:

  • Different organisms (plants vs. mammals)

  • Different cell types (expression levels vary across tissues)

  • Different experimental systems (in vitro vs. in vivo)

• Methodological considerations:

  • Antibody specificity issues - verify with appropriate controls

  • Splice variant detection - ensure your experimental system can distinguish relevant isoforms

  • Knockout/knockdown efficiency - incomplete silencing may yield inconsistent results

• Integrated analysis approach:

  • Combine multiple methodologies (e.g., genetic, biochemical, and cellular approaches)

  • Consider temporal factors, especially when studying plant PRR5 which shows circadian expression patterns

  • Analyze PRR5 in conjunction with interaction partners to understand context-specific functions

• Genetic interaction analysis:

  • For plant research, consider using combinations of mutants (e.g., prr5 prr7, prr9 prr7, prr5 prr7 abi5 triple mutant)

  • Functional redundancy may mask phenotypes in single mutants

• Resolution strategies:

  • Replicate experiments using standardized conditions

  • Consider temporal factors in experimental design, particularly for circadian proteins

  • Validate findings using complementary approaches (e.g., protein-level and genetic studies)

What are the optimal sample preparation methods for PRR5 detection?

For consistent and reliable PRR5 detection across different experimental approaches:

Western Blot sample preparation:

• Use RIPA or NP-40 based lysis buffers supplemented with protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation status

• For mammalian PRR5, HepG2, Jurkat, and MCF-7 cell lines consistently show good expression

• For plant PRR5, extraction from tissues entrained in circadian cycles should consider timing of collection, as PRR5 levels show diurnal variation

Immunohistochemistry sample preparation:

• For optimal antigen retrieval, use TE buffer at pH 9.0

• Alternative protocol: citrate buffer at pH 6.0

• Fixation with 4% paraformaldehyde works well for most tissues

Immunofluorescence optimization:

• Starting dilution of 1:10-1:100 is recommended

• Permeabilization with 0.1-0.5% Triton X-100 improves intracellular detection

• For mammalian PRR5, HepG2 cells have been validated for IF detection

Time-dependent considerations for plant PRR5:

• Under long-day conditions, PRR5 protein peaks at dusk despite undetectable mRNA levels

• Dark transition induces rapid PRR5 degradation through ZTL-mediated pathways

• Collection timing is critical - protein levels may not correlate with mRNA abundance due to post-translational regulation

How can I optimize ChIP experiments for studying PRR5 (plant) DNA binding?

ChIP experiments for plant PRR5 require specific considerations:

• Sample collection timing:

  • PRR5 binding to target DNA follows a circadian pattern

  • Optimal timing for ChIP sample collection varies by target gene

  • For CCA1/LHY targets, binding peaks during midday to evening hours

• Crosslinking and sonication:

  • Standard 1% formaldehyde crosslinking for 10-15 minutes is effective

  • Sonication conditions should be optimized to yield DNA fragments of 200-500 bp

• Antibody selection:

  • Use ChIP-validated antibodies or epitope-tagged PRR5 constructs

  • PRR5pro:FLAG-PRR5-GFP in a prr5 mutant background has been successfully used

• Positive controls:

  • Include known PRR5 targets like CCA1 and LHY promoter regions

  • Validate enrichment using qPCR before proceeding to genome-wide approaches

• Data analysis considerations:

  • PRR5 shows enrichment at upstream regions of target genes

  • Focus analysis on promoter regions within 1kb of transcription start sites

  • For ChIP-seq, use input DNA as control and apply appropriate FDR cutoffs (q < 10^-50 has been used)

• Validation of binding functionality:

  • Combine ChIP data with expression analysis to correlate binding with transcriptional effects

  • Consider using transient expression assays with CCT domain mutants as functional validation

What are the best approaches for generating PRR5 expression constructs?

When designing PRR5 expression constructs, consider domain-specific functions and experimental requirements:

For mammalian PRR5 constructs:

• Full-length human PRR5 is 388 amino acids (GenBank BC016921)

• Consider using codon-optimized sequences for improved expression

• For GST-fusion proteins, pGEX-6P-1 or pEBG vectors have been successfully used

• When studying splice variants, be aware of alternate start sites (Met10, Met96) and internal deletions

For plant PRR5 constructs:

• Domain-specific constructs should consider functional regions:

  • PR domain (aa 1-180): involved in ZTL interaction

  • C-terminal fragment (aa 172-558): critical for ABI5 interaction

  • CCT domain (aa 502-558): essential for DNA binding

• For transcriptional studies, VP16 fusion constructs can assess DNA association

• Point mutations in the CCT domain (e.g., Ala538Val or Arg543His) can serve as negative controls

Tagging strategies:

• C-terminal tags are generally preferred to avoid interfering with N-terminal domains

• Successfully used tags include:

  • FLAG tag for immunoprecipitation and ChIP studies

  • GFP for localization and live-cell imaging

  • GST for pull-down assays

  • VP16 for transcriptional activity assays

  • Glucocorticoid receptor (GR) fusion for inducible nuclear localization

Verification methods:

• Sequence verification of all constructs is essential

• Western blot validation of expression and correct size

• Functional validation through complementation of knockout/knockdown systems

How should I design experiments to study the circadian regulation of plant PRR5?

When studying plant PRR5 circadian patterns:

• Entrainment conditions:

  • Synchronize plants with consistent light/dark cycles (typically 12L/12D) for at least 7 days

  • For free-running experiments, transfer to constant light or dark after entrainment

  • Document zeitgeber time (ZT) or circadian time (CT) precisely in all experiments

• Time-course sampling:

  • Collect samples every 3-4 hours over a 24-hour period for basic profiling

  • For higher resolution, 2-hour intervals are recommended

  • Include at least one complete circadian cycle (24 hours)

• PRR5 protein vs. mRNA measurements:

  • Be aware that protein levels lag behind mRNA expression

  • PRR5 protein persists after mRNA levels decline, particularly at dusk

  • Dark-induced degradation occurs rapidly through ZTL-mediated pathways

• Studying PRR5 targets:

  • PRR5 binding to target genes follows a circadian pattern

  • Direct targets show suppressed expression when PRR5 is bound to their promoters

  • Key targets include CCA1 and LHY, which should be monitored along with PRR5

• Mutant analysis:

  • Single prr5 mutants may show subtle phenotypes due to functional redundancy with PRR7 and PRR9

  • Higher-order mutants (prr5 prr7, prr9 prr7) often show more pronounced phenotypes

  • For genetic interaction studies, include appropriate combinations (e.g., prr5 prr7 abi5)

What methodological approaches are recommended for studying PRR5's role in mTORC2 signaling?

For investigating mammalian PRR5's function in mTORC2:

• Protein complex analysis:

  • Co-immunoprecipitation to assess PRR5 interaction with mTORC2 components

  • Size-exclusion chromatography to analyze intact complex formation

  • Two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) has been successfully used to identify PRR5 as an mTOR interactor

• Functional studies:

  • siRNA knockdown of PRR5 to assess effects on mTORC2 assembly and function

  • Overexpression of wild-type and domain mutants of PRR5

  • Analysis of downstream targets of mTORC2 (e.g., Akt, PKC alpha phosphorylation)

• Domain-specific experiments:

  • Focus on the two RICTOR interaction sites (aa 10-95 and 188-218)

  • Generate deletion constructs to map functional domains

  • Co-expression with SIN1 siRNA to assess dependency of mTOR binding

• Cell-based assays:

  • HEK293 cells provide a good model system for studying PRR5-mTORC2 interactions

  • Consider cell type-specific effects as PRR5 function may vary across tissues

  • Include appropriate controls (e.g., raptor knockdown, which should not affect PRR5-mTOR binding)

• Readout assays:

  • Phosphorylation of Akt at Ser473 as a marker of mTORC2 activity

  • Cell growth and survival assays to assess functional outcomes

  • Apoptosis assays as PRR5 may regulate cell survival through mTORC2

What controls are essential when performing immunohistochemistry with PRR5 antibodies?

When performing IHC with PRR5 antibodies, include these critical controls:

• Negative controls:

  • Omission of primary antibody

  • Isotype control (rabbit IgG for most PRR5 antibodies)

  • Tissue from PRR5 knockout/knockdown models

  • Pre-absorption of antibody with immunizing peptide

• Positive controls:

  • Validated tissue types with known PRR5 expression:

    • Human: kidney, brain, placenta tissues

    • Mouse: brain, testis, kidney tissues

  • Cells with confirmed PRR5 expression (e.g., HepG2, HEK-293)

• Antigen retrieval optimization:

  • Compare TE buffer pH 9.0 (recommended) with citrate buffer pH 6.0

  • Titrate antibody dilution (starting range: 1:50-1:500)

  • Include time and temperature controls for antigen retrieval

• Specificity controls:

  • Western blot correlation to verify specificity before IHC

  • Multiple antibodies targeting different epitopes of PRR5

  • Fluorescent double-labeling with known markers of PRR5-expressing cells

• Technical controls:

  • Include positive control for the detection system

  • Standardize all washing steps and incubation times

  • Document all methodology details for reproducibility