PER22 Antibody

What are the validated applications for PER2 antibody?

PER2 antibody (such as 67513-1-Ig) has been validated for several experimental applications in research settings. Based on extensive testing, the primary applications include:

• Western Blot (WB): Successfully used in at least 11 published studies

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Validated in cellular models

• Immunohistochemistry (IHC): Validated in at least 2 published studies

• ELISA: Successfully employed for protein detection

The antibody shows strong reactivity with human samples and has cited reactivity with mouse and pig samples as well . When designing your experiments, it's important to verify the specific validation data for your particular experimental system.

What are the recommended dilutions for different experimental applications of PER2 antibody?

Optimal dilution of antibodies is critical for obtaining specific signals while minimizing background. For PER2 antibody (67513-1-Ig), the following dilutions are recommended based on empirical testing:

It is important to note that these ranges should be considered starting points, and optimization is recommended for each specific experimental system. As indicated in antibody guidelines, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and results may be "Sample-dependent" .

How should PER2 antibody be stored to maintain optimal activity?

Proper storage of antibodies is essential for maintaining their activity and specificity. For PER2 antibody:

• Store at -20°C for long-term stability

• The antibody is stable for one year after shipment when stored properly

• Aliquoting is unnecessary for -20°C storage

• The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

Smaller size antibody preparations (e.g., 20μl) may contain 0.1% BSA as a stabilizing agent. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity .

What controls should be included when using PER2 antibody in experiments?

Including appropriate controls is essential for antibody validation and experimental reproducibility. Based on best practices in antibody research, the following controls should be incorporated:

For PER2 antibody, positive WB detection has been validated in HEK-293, L02, Y79, BxPC-3, K-562, HL-60, and THP-1 cells, while positive IF/ICC has been confirmed in HeLa cells .

How can I validate a PER2 antibody for novel experimental applications?

Validating antibodies for new applications requires a systematic approach:

• Cross-referencing validation data: Review the full blot data provided by the manufacturer and published literature. For PER2 antibody, check the validation data gallery available from the supplier .

• Specificity testing: For novel applications, run validation controls including:

  • Knockout/knockdown samples as negative controls

  • Overexpression systems as positive controls

  • Peptide competition assays to confirm binding specificity

• Dilution optimization: Perform a dilution series experiment using a range of antibody concentrations and protein loads. For PER2 antibody, start with the recommended dilutions (WB: 1:5000-1:50000; IF/ICC: 1:400-1:1600) and adjust as needed .

• Cross-validation: Compare results with alternative methods (e.g., mass spectrometry, alternative antibodies targeting different epitopes) .

When publishing research using PER2 antibody in novel applications, provide detailed validation data following the guidelines outlined in physiology journals, including representative full blots demonstrating specificity .

What factors affect the observed molecular weight of PER2 in western blot analysis?

Researchers may encounter discrepancies between calculated and observed molecular weights when working with PER2 antibody:

• Calculated molecular weight: 137 kDa

• Observed molecular weight: 150-160 kDa

This discrepancy can be attributed to several factors:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can increase apparent molecular weight

• Splice variants: Different isoforms may show distinct banding patterns

• Buffer conditions and sample preparation: Denaturation methods can affect protein migration

• Gel percentage and running conditions: These parameters can influence protein migration patterns

When analyzing western blot results with PER2 antibody, document both the expected theoretical weight and the empirically observed weight, noting any conditions that might affect migration patterns .

How should I approach contradictory results between different detection methods using PER2 antibody?

When facing contradictory results between different detection methods (e.g., discrepancies between WB and IHC results), consider these methodological approaches:

• Epitope accessibility: The PER2 epitope may be differentially accessible in various techniques due to protein conformation, fixation methods, or protein-protein interactions

• Protocol optimization: Each technique requires specific optimization:

  • For WB: Adjust extraction buffers, denaturation conditions, and blocking agents

  • For IHC/IF: Test different fixation methods, antigen retrieval protocols, and incubation conditions

• Validation approach: Implement a multi-technique validation strategy:

  • Use multiple antibodies targeting different epitopes of PER2

  • Compare results with genetic approaches (siRNA knockdown or CRISPR knockout)

  • Consider orthogonal methods like mass spectrometry

• Careful documentation: Record all experimental conditions meticulously, including antibody details (lot number, dilution), sample preparation methods, and image acquisition parameters

What considerations are important when designing longitudinal studies measuring PER2 antibody responses?

When designing longitudinal studies to monitor antibody responses over time (similar to the approach described in study ), several methodological considerations are crucial:

• Sampling intervals: Establish appropriate time points based on expected kinetics of the response. In antibody response studies, intervals of 3-6 months have been used to track changes .

• Consistent methodology: Maintain identical experimental conditions throughout the study:

  • Use the same antibody lot when possible

  • Standardize sample collection, processing, and storage protocols

  • Include internal controls on each experimental run

• Data normalization: Implement appropriate normalization strategies to account for inter-assay variability:

  • Include standard curves on each experimental run

  • Use internal control samples across different time points

  • Consider statistical approaches to account for batch effects

• Participant/sample tracking: Develop robust systems for tracking samples and associated metadata over time, including detailed questionnaires to document relevant exposures or interventions .

How can I optimize PER2 antibody for cell type-specific applications?

Optimizing PER2 antibody performance across different cell types requires systematic adaptation of protocols:

• Cell-specific validation: PER2 antibody (67513-1-Ig) has been validated in multiple cell lines, including HEK-293, L02, Y79, BxPC-3, K-562, HL-60, THP-1, and HeLa cells . When working with new cell types:

  • Start with the recommended dilutions

  • Perform a validation series with positive and negative controls

  • Optimize fixation methods (for IF/ICC) or lysis buffers (for WB)

• Expression level considerations: PER2 expression varies across cell types and may be affected by circadian rhythm. Consider:

  • Timing of sample collection

  • Cell synchronization methods

  • Protein loading amounts for western blot

• Background reduction strategies:

  • Optimize blocking conditions (concentration, type of blocking agent)

  • Adjust antibody incubation time and temperature

  • Test various washing protocols to improve signal-to-noise ratio

What are the best approaches for quantifying PER2 expression in tissue samples?

Quantifying PER2 expression in tissue samples requires careful consideration of several methodological factors:

• Sample preparation standardization:

  • Consistent tissue collection and processing protocols

  • Standardized fixation methods and times

  • Uniform sectioning thickness for tissue samples

• Immunohistochemistry optimization:

  • Antigen retrieval methods should be systematically tested

  • Antibody concentration should be titrated for each tissue type

  • Incubation conditions (time, temperature) should be optimized

• Quantification methods:

  • Define clear scoring systems (e.g., H-score, percentage positive cells)

  • Use digital image analysis for objective quantification

  • Include appropriate positive and negative controls in each batch

  • Compare results with other detection methods (e.g., western blot, RT-PCR)

• Data analysis considerations:

  • Account for tissue heterogeneity

  • Consider spatial distribution of staining

  • Use appropriate statistical methods for comparative analyses

How can I minimize cross-reactivity when using PER2 antibody in multiplex immunoassays?

When incorporating PER2 antibody into multiplex immunoassays, minimizing cross-reactivity is essential:

• Antibody selection: Choose antibodies from different host species when possible or use isotype-specific secondary antibodies. For PER2 antibody (67513-1-Ig), note that it is a Mouse IgG1 monoclonal antibody .

• Sequential staining protocols:

  • Consider sequential rather than simultaneous incubation

  • Block between sequential antibody applications

  • Use direct conjugates when possible to avoid secondary antibody cross-reactivity

• Validation controls:

  • Single antibody controls to establish baseline signals

  • Isotype controls to detect non-specific binding

  • Absorption controls with competing antigens

• Signal separation strategies:

  • Optimize fluorophore selection to minimize spectral overlap

  • Implement computational approaches for spectral unmixing

  • Consider spatial separation of antigens being detected

What are the considerations for using PER2 antibody in circadian rhythm research?

PER2 (Period homolog 2) is a critical component of the circadian clock mechanism, requiring specific experimental considerations:

• Temporal sampling strategy:

  • Design sampling protocols that account for circadian oscillations

  • Collect samples at multiple time points over a 24-hour cycle

  • Document timing relative to synchronizing zeitgebers

• Synchronization protocols:

  • Standardize methods to synchronize cellular circadian rhythms

  • Document entrainment conditions (light/dark cycles, feeding schedules)

  • Consider the impact of synchronization methods on antibody epitope accessibility

• Data analysis approaches:

  • Implement time series analysis methods

  • Consider circadian phase and amplitude as key parameters

  • Compare PER2 expression patterns with other clock proteins

• Technical considerations:

  • Observed molecular weight of PER2 (150-160 kDa) may vary depending on phosphorylation state throughout the circadian cycle

  • Subcellular localization changes may require optimization of extraction methods

How can PER2 antibody be utilized in high-avidity, low-affinity (HALA) antibody research?

Although the search results don't specifically address PER2 antibody in HALA research, general principles from antibody engineering research can be applied:

• Mechanistic modeling: Computational models can predict how antibody binding kinetics affect tissue distribution and efficacy:

  • Evaluate competitive binding between different antibody formats

  • Consider the impact of receptor density on binding dynamics

  • Model diffusion rates in different tissue environments

• Experimental validation approaches:

  • Compare standard PER2 antibody with engineered variants

  • Assess binding competition through displacement assays

  • Evaluate tissue penetration in spheroid models

• Analytical considerations:

  • Develop appropriate binding kinetics assays

  • Implement imaging approaches to visualize tissue distribution

  • Consider dimensional analysis to create predictive models

What quality control measures should be implemented when validating new lots of PER2 antibody?

Consistent antibody performance across different lots is critical for research reproducibility. Based on guidelines for antibody use in physiology studies , researchers should implement these quality control measures:

• Lot-to-lot comparison testing:

  • Run side-by-side western blots with old and new antibody lots

  • Compare staining patterns in immunohistochemistry/immunofluorescence

  • Document any differences in sensitivity or specificity

• Standardized validation protocol:

  • Maintain a consistent protocol for validating each new lot

  • Include positive and negative controls

  • Test across a range of dilutions to assess potency

• Documentation requirements:

  • Record detailed antibody information (Fig. 2 from source )

  • Maintain a validation database comparing lot performance

  • Document any adjustments needed for new lots

• Performance metrics:

  • Signal-to-noise ratio comparisons

  • Dilution curve analysis

  • Target specificity assessment

What information should be included when reporting PER2 antibody use in publications?

Based on guidelines for antibody use in physiology studies , comprehensive reporting should include:

• Antibody identification:

  • Complete source information (manufacturer, catalog number)

  • Clone designation for monoclonal antibodies

  • Host species and isotype (e.g., Mouse IgG1 for 67513-1-Ig)

  • RRID (Research Resource Identifier) when available (e.g., AB_2882734)

• Validation documentation:

  • Reference to prior validation or new validation data

  • Representative full blots as supplementary material

  • Controls used to confirm specificity

  • Explanation of any unexpected banding patterns

• Experimental conditions:

  • Detailed protocol with antibody dilution (e.g., 1:5000-1:50000 for WB)

  • Sample preparation methods

  • Detection systems employed

  • Image acquisition parameters

  • Quantification methodology

• Reproducibility considerations:

  • Number of experimental replicates

  • Consistency across different samples

  • Statistical approaches for data analysis

Following these reporting standards enhances research reproducibility and aligns with journal requirements for antibody-based studies .

How can HAHA (Human Anti-Human Antibody) assays inform PER2 antibody experimental design?

While HAHA assays described in source are primarily relevant for therapeutic antibodies, the methodological principles can inform research antibody validation:

• Cross-reactivity testing:

  • Design competitive ELISA assays similar to HAHA testing to evaluate antibody specificity

  • Use anti-idiotype antibodies as controls for binding specificity

  • Implement dilution series to establish dose-response relationships

• Validation methodology:

  • Establish standard curves with varying dilutions

  • Run samples in triplicate to assess reproducibility

  • Define acceptance criteria for valid assays based on linear regression parameters

• Interference assessment:

  • Test for potential interfering substances in complex biological samples

  • Evaluate the impact of sample matrix on antibody performance

  • Consider pre-absorption steps to reduce non-specific binding

These approaches can enhance the rigor of PER2 antibody validation and provide more reliable experimental results .