PER3 Antibody, Biotin conjugated

What is PER3 protein and why is it significant in research?

PER3 (Period Circadian Protein Homolog 3) is a critical component of the circadian clock system that functions as a negative regulator of the circadian rhythm. This protein plays essential roles in regulating sleep-wake cycles, hormone secretion, metabolism, and other physiological processes. PER3 is also involved in cell proliferation, apoptosis, DNA damage response, cell cycle control, and chemotherapy treatment response . The significance of PER3 in research stems from its implications in circadian rhythm disorders, sleep timing differences, and potential connections to certain cancer types. Variations in the PER3 gene can lead to differences in circadian rhythm function, making it a valuable target for studies on sleep disorders and chronobiology .

What are the structural and technical specifications of biotin-conjugated PER3 antibody?

The biotin-conjugated PER3 antibody is typically a polyclonal antibody generated in rabbits by immunization with recombinant human PER3 protein (amino acids 1-131) . The antibody undergoes purification through protein G affinity chromatography, achieving purity levels exceeding 95% . It is supplied in liquid form in a buffer containing stabilizers such as PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol . The antibody specifically targets human PER3 protein and is validated for applications including ELISA . The molecular weight of the target PER3 protein is approximately 132 kDa, consisting of 1201 amino acids .

What are the optimal storage conditions for preserving antibody activity?

For maximum stability and activity preservation, the biotin-conjugated PER3 antibody should be stored at -20°C . The antibody should be aliquoted before freezing to minimize freeze-thaw cycles, which can degrade protein quality and compromise functionality . Exposure to light should be minimized as biotin conjugation can be photosensitive . The product is typically stable for one year after shipment when stored properly . For smaller volumes (20μl sizes), some manufacturers indicate that aliquoting may be unnecessary for -20°C storage, particularly when the solution contains stabilizing agents such as BSA . Researchers should note that improper storage can lead to reduced signal strength in applications and potential false-negative results.

What applications is the biotin-conjugated PER3 antibody validated for?

What methodological considerations are important when using biotin-conjugated antibodies in research protocols?

When utilizing biotin-conjugated PER3 antibody, researchers should consider several methodological aspects to ensure experimental success:

• Avidin-Biotin Interaction: Biotin-conjugated antibodies can be detected using avidin, streptavidin, or neutravidin conjugated to reporter molecules (enzymes, fluorophores). This interaction has extremely high affinity and provides signal amplification .

• Endogenous Biotin Blocking: Tissues like liver, kidney, and brain contain endogenous biotin that can cause background signal. Pre-block with unconjugated avidin/streptavidin or use commercial biotin-blocking kits.

• Dilution Optimization: Though manufacturers provide recommended dilution ranges, optimal concentrations should be empirically determined for each specific experimental system . Start with the suggested range and adjust based on signal-to-noise ratio.

• Positive Controls: When performing experiments with PER3 antibody, include validated positive controls such as HeLa cells, which have been confirmed to express detectable levels of PER3 .

• Signal Development Time: When using biotin-conjugated antibodies in enzymatic detection systems, carefully monitor the development time to prevent oversaturation while maintaining sensitivity.

How should I design appropriate controls when using PER3 antibody, biotin conjugated?

Proper control design is essential for ensuring the validity and reliability of experiments using biotin-conjugated PER3 antibody:

• Positive Controls: Include samples known to express PER3, such as HeLa cells for human PER3 detection . Positive controls verify that the detection system is functioning correctly.

• Negative Controls: Use samples where PER3 expression is absent or has been knocked down. Additionally, perform experiments omitting the primary antibody to assess secondary detection system specificity.

• Isotype Controls: Include a biotin-conjugated rabbit IgG isotype control at the same concentration as the PER3 antibody to identify nonspecific binding.

• Blocking Controls: For biotin-conjugated antibodies, include controls to evaluate the effectiveness of endogenous biotin blocking to confirm that signals are specific to PER3 detection rather than endogenous biotin.

• Antibody Concentration Gradient: Perform a titration of antibody concentrations to determine the optimal signal-to-noise ratio for your specific samples and detection system.

What are common sources of false results when using biotin-conjugated PER3 antibody, and how can they be mitigated?

Several factors can contribute to false results when working with biotin-conjugated PER3 antibody:

• Endogenous Biotin Interference: Tissues and cells can contain natural biotin, leading to false-positive signals in detection systems utilizing avidin-biotin interactions. This can be mitigated by:

  • Implementing a biotin-blocking step prior to antibody application

  • Using specialized biotin-blocking kits

  • Considering alternative detection methods for tissues known to be biotin-rich

• Antibody Degradation: Loss of antibody activity due to improper storage or excessive freeze-thaw cycles can produce false-negative results. Prevention strategies include:

  • Storing the antibody strictly at -20°C

  • Aliquoting the antibody upon receipt to minimize freeze-thaw cycles

  • Avoiding exposure to light, which can affect the biotin conjugate

• Cross-Reactivity: While the PER3 antibody is designed to be specific, structural similarities between proteins can lead to cross-reactivity and false-positive signals. Address this by:

  • Conducting validation tests using PER3-knockout or knockdown samples

  • Including appropriate negative controls

  • Verifying results with an alternative antibody targeting a different epitope of PER3

• Insufficient Blocking: Inadequate blocking can result in high background and false-positive signals. Improve blocking by:

  • Optimizing blocking buffer composition

  • Extending blocking time

  • Using casein-based blockers, which can be more effective with biotin-conjugated detection systems

How can I optimize signal-to-noise ratio when using biotin-conjugated PER3 antibody?

Optimizing signal-to-noise ratio requires methodical adjustments to experimental parameters:

• Antibody Concentration Titration: Test a range of antibody dilutions to identify the concentration that provides maximal specific signal with minimal background. Typical dilution ranges for PER3 antibody applications include 1:500-1:5000 for WB and 1:20-1:200 for IHC, though these should be adjusted for the biotin-conjugated version .

• Incubation Conditions: Optimize temperature and duration of antibody incubation. For biotin-conjugated antibodies, overnight incubation at 4°C often produces better signal-to-noise ratio than shorter incubations at room temperature.

• Washing Protocols: Increase washing stringency by:

  • Adding detergents like Tween-20 at 0.05-0.1% to washing buffers

  • Increasing the number and duration of washing steps

  • Using automated washers for consistent washing efficiency

• Detection System Optimization: When using avidin-HRP or streptavidin-HRP systems:

  • Titrate the concentration of the detection reagent

  • Consider using enhanced chemiluminescence substrates for Western blots

  • For IHC/IF applications, use tyramide signal amplification for enhanced sensitivity while maintaining low background

• Sample Preparation: Improve sample quality by:

  • Using fresh or properly preserved samples

  • Optimizing fixation conditions

  • Implementing appropriate antigen retrieval methods for IHC applications, such as TE buffer pH 9.0 which has been recommended for PER3 detection

How should I interpret differences in PER3 protein detection patterns across experimental conditions?

Interpretation of PER3 detection patterns requires consideration of biological and technical factors:

• Circadian Variation: PER3 expression follows circadian rhythms, so variations in detection may reflect time-of-day differences in sample collection. Document sampling time and consider time-course experiments to capture circadian expression patterns.

• Molecular Weight Verification: The expected molecular weight of PER3 is approximately 132 kDa . Bands at different molecular weights may indicate:

  • Post-translational modifications

  • Alternative splice variants

  • Proteolytic degradation

  • Non-specific binding

• Subcellular Localization: PER3 shuttles between cytoplasm and nucleus in a circadian-dependent manner. Differences in subcellular localization patterns may reflect:

  • Different phases of the circadian cycle

  • Disrupted nuclear transport

  • Altered protein-protein interactions affecting localization

• Expression Level Variations: Quantitative differences in PER3 detection across samples may indicate:

  • Genetic variations affecting expression levels

  • Pathological conditions altering circadian gene expression

  • Environmental factors (light exposure, feeding schedules) impacting circadian regulation

  • Treatment effects on circadian pathway components

• Technical Considerations: Variation in detection may also result from technical issues:

  • Sample preparation inconsistencies

  • Antibody concentration differences

  • Detection system variations

  • Buffer composition effects on epitope accessibility

How can PER3 antibody, biotin conjugated be utilized in circadian rhythm research?

The biotin-conjugated PER3 antibody offers several advanced applications in circadian rhythm research:

• Circadian Protein Oscillation Profiling: The antibody can be used in time-course experiments to monitor PER3 protein oscillations across the 24-hour cycle in various tissues and cell types. The biotin conjugation provides signal amplification advantages for detecting subtle changes in protein levels.

• Chromatin Immunoprecipitation Sequential (ChIP-seq) Studies: For investigating PER3's role in transcriptional regulation, biotin-conjugated antibodies offer advantages in ChIP-seq protocols due to the high-affinity binding between biotin and streptavidin, allowing for efficient pull-down of protein-DNA complexes.

• Protein-Protein Interaction Studies: The biotin tag enables techniques such as:

  • Pull-down assays using streptavidin beads to identify PER3 interaction partners

  • Proximity-dependent biotin identification (BioID) to map the PER3 protein interaction network

  • Förster Resonance Energy Transfer (FRET) assays using biotin-streptavidin systems to study dynamic protein interactions in living cells

• Single-Cell Protein Quantification: Using biotin-conjugated PER3 antibody with flow cytometry or mass cytometry allows for analysis of heterogeneity in PER3 expression at the single-cell level, providing insights into cell-to-cell variability in circadian rhythm components.

• Circadian Rhythm Disruption Models: The antibody can be used to investigate the effects of sleep deprivation, shift work, or jet lag models on PER3 expression and localization, contributing to understanding the molecular basis of circadian disruption-related disorders.

What methodological approaches can be used to study PER3's role in cancer research?

PER3 has been implicated in cancer biology through its roles in cell cycle regulation, DNA damage response, and apoptosis . Advanced methodological approaches using biotin-conjugated PER3 antibody include:

• Tissue Microarray Analysis: This approach allows for high-throughput screening of PER3 expression across multiple cancer samples and matched normal tissues. The biotin-conjugated antibody provides signal amplification benefits for detecting expression differences.

• Co-localization Studies: Implement dual immunofluorescence labeling to investigate:

  • PER3 co-localization with cell cycle regulators

  • Association with DNA damage markers

  • Interaction with apoptotic pathway components

• Post-Translational Modification Profiling: Combine PER3 detection with phospho-specific antibodies to assess how cancer-related signaling pathways affect PER3 phosphorylation status, which regulates its stability and function.

• Circadian Rhythm Disruption in Cancer Models: Utilize the antibody to examine how disrupted circadian rhythms in cancer models affect PER3 expression patterns and whether restoration of normal PER3 cycling impacts cancer progression.

• Therapeutic Response Monitoring: Evaluate changes in PER3 expression and localization in response to cancer therapeutics, particularly chronotherapy approaches that time treatment administration based on circadian rhythms.

How does PER3 antibody detection correlate with genetic variations in the PER3 gene?

Research into PER3 genetic variations, particularly the variable number tandem repeat (VNTR) polymorphism, has revealed associations with sleep preferences, cognitive performance, and disease susceptibility. The biotin-conjugated PER3 antibody can be employed to investigate protein-level consequences of these genetic variations:

• Protein Expression Quantification: Use quantitative immunoassays to compare PER3 protein levels across samples with different PER3 genotypes, correlating genetic variations with protein expression differences.

• Isoform-Specific Detection: The PER3 gene produces multiple transcript variants. Carefully designed epitope mapping can help determine which protein isoforms are detected by the antibody, providing insights into isoform-specific functions.

• Functional Correlation Studies: Combine genotyping for PER3 polymorphisms with protein detection to establish relationships between:

  • PER3 variants and protein half-life

  • Genetic variations and subcellular localization patterns

  • Polymorphisms and protein-protein interaction profiles

• Differential Response to Environmental Cues: Investigate how cells with different PER3 genotypes respond to synchronizing stimuli (e.g., serum shock, temperature cycles) by monitoring PER3 protein dynamics using the biotin-conjugated antibody.

• Clinical Correlation Studies: In human studies, correlate PER3 genotype with protein expression in accessible tissues (e.g., buccal cells, fibroblasts) to understand the translation of genetic variations to protein-level phenotypes.

How can I validate the specificity of PER3 antibody, biotin conjugated for my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For PER3 antibody, biotin conjugated, consider these approaches:

• Genetic Knockdown/Knockout Controls: Using siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate PER3 expression provides definitive evidence of antibody specificity. A proportional reduction in signal intensity should be observed in knockdown samples.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (PER3 amino acids 1-131) before application to samples. Specific binding should be blocked, resulting in signal reduction or elimination.

• Cross-Species Reactivity Testing: The antibody is reported to react with human PER3, with some products also reacting with mouse and rat samples . Testing across species can help confirm target specificity and evolutionary conservation.

• Comparison with Alternative Antibodies: Compare detection patterns with other validated PER3 antibodies targeting different epitopes. Concordant results increase confidence in specificity.

• Mass Spectrometry Validation: For definitive validation, perform immunoprecipitation with the PER3 antibody followed by mass spectrometry analysis to confirm the identity of the precipitated proteins.

What advanced detection systems can enhance the sensitivity of biotin-conjugated PER3 antibody?

Several advanced detection systems can be employed to maximize sensitivity when using biotin-conjugated PER3 antibody:

• Tyramide Signal Amplification (TSA): This enzyme-mediated detection method can increase sensitivity by 10-100 fold compared to conventional detection. The horseradish peroxidase (HRP) conjugated to streptavidin catalyzes the deposition of multiple tyramide-fluorophore or tyramide-chromogen molecules, creating signal amplification.

• Quantum Dot (QD) Conjugation: Streptavidin-conjugated quantum dots provide advantages including:

  • Exceptional photostability compared to organic fluorophores

  • Narrow emission spectra allowing for multiplexing

  • Higher sensitivity due to bright fluorescence

• Rolling Circle Amplification (RCA): This isothermal enzymatic process can amplify detection signals by generating thousands of copies of a circular DNA template, which can then be detected with fluorescent probes.

• Proximity Ligation Assay (PLA): For co-localization studies, combining biotin-conjugated PER3 antibody with another primary antibody in PLA format allows detection of proteins in close proximity (<40 nm), providing insights into protein-protein interactions.

• Single-Molecule Detection Systems: Advanced microscopy techniques like total internal reflection fluorescence (TIRF) or stochastic optical reconstruction microscopy (STORM) can detect biotin-conjugated antibodies at the single-molecule level, offering unprecedented sensitivity.

How can I adapt protocols for simultaneous detection of PER3 and other circadian clock proteins?

Investigating the circadian clock system often requires simultaneous detection of multiple components. To adapt protocols for multiplexed detection including PER3:

• Antibody Compatibility Assessment: Ensure primary antibodies are raised in different host species to allow species-specific secondary detection. For example, use rabbit anti-PER3 biotin conjugated with mouse anti-CLOCK or mouse anti-BMAL1 antibodies.

• Sequential Immunostaining: For tissues with high autofluorescence or when antibodies are from the same species:

  • Perform complete detection of PER3 first

  • Follow with chemical stripping of the first set of antibodies while preserving tissue architecture

  • Proceed with detection of secondary targets

• Spectral Separation Optimization: When designing fluorescent multiplexing:

  • Choose fluorophores with minimal spectral overlap

  • Utilize biotin-streptavidin systems with far-red fluorophores for PER3

  • Apply appropriate controls for spectral bleed-through

• Differential Subcellular Localization Analysis: Exploit the fact that circadian proteins show time-dependent nuclear-cytoplasmic shuttling by using nuclear and cytoplasmic markers in conjunction with PER3 antibody to track relative localizations.

• Advanced Imaging Analysis: Implement software-based analysis tools that can:

  • Quantify co-localization coefficients

  • Perform object-based colocalization analysis

  • Generate proximity maps between PER3 and other clock proteins

  • Analyze temporal dynamics of protein interactions

By applying these advanced methodologies, researchers can gain deeper insights into the complex interactions and dynamics of PER3 within the circadian clock system and related cellular processes.