PCR8 Antibody

What is PAQR8 and why is it a target for antibody development?

PAQR8 (Progestin and adipoQ receptor family member 8), also known as membrane progestin receptor beta (mPR beta), is a receptor protein that may be involved in oocyte maturation. Research indicates that PAQR8 binds several steroid hormones including dehydroepiandrosterone (DHEA), pregnanolone, pregnenolone, and allopregnanolone . As a membrane receptor with potential roles in hormone signaling, PAQR8 represents an important target for antibody development to study its cellular localization, protein interactions, and functional roles in reproductive biology and other physiological processes.

What types of PAQR8 antibodies are available for research applications?

Current research applications primarily utilize polyclonal antibodies targeting specific regions of PAQR8. For example, commercially available antibodies include affinity-purified rabbit polyclonal antibodies that target the C-terminal region (amino acids 326-354) of human PAQR8 . These antibodies typically demonstrate reactivity against both human and mouse PAQR8, making them suitable for cross-species studies. While most available antibodies are polyclonal, the development of monoclonal antibodies against PAQR8 would follow similar methodological approaches as those used for other membrane receptors, such as the epitope mapping strategies employed for CCR8 antibodies .

What applications are PAQR8 antibodies validated for?

Based on current validation data, PAQR8 antibodies are primarily validated for Western blot (WB) applications with a recommended dilution of 1:1000 . While the primary application is Western blotting, researchers should consider validating these antibodies for additional applications such as immunohistochemistry, immunofluorescence, or immunoprecipitation depending on their specific research needs. Validation across multiple applications would follow similar methodological approaches as those used for other antibodies in the field.

How should researchers validate PAQR8 antibodies before experimental use?

A comprehensive validation approach should include:

• Positive and negative controls: Using tissues or cell lines known to express or not express PAQR8

• Knockout/knockdown validation: Testing antibody specificity using PAQR8 knockout or knockdown samples

• Cross-reactivity testing: Evaluating potential cross-reactivity with related PAQR family members

• Application-specific validation: Confirming antibody performance in each intended application

• Lot-to-lot consistency testing: Comparing performance between different antibody lots

This validation approach aligns with general best practices in antibody research that emphasize rigorous specificity testing before experimental application .

What experimental controls are essential when using PAQR8 antibodies?

These controls are essential for meaningful interpretation of experimental results and follow standard methodological approaches in antibody-based research .

What are the optimal storage conditions for maintaining PAQR8 antibody efficacy?

PAQR8 antibodies are typically supplied in PBS with 0.09% (W/V) sodium azide . For optimal longevity and performance:

• Store antibody aliquots at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing smaller working aliquots

• For short-term storage (1-2 weeks), antibodies can be kept at 4°C

• Include carrier proteins (e.g., BSA) for diluted antibody solutions

• Monitor antibody performance over time with consistent positive controls

These storage recommendations align with general antibody preservation protocols to maintain binding efficacy and specificity.

How can researchers perform epitope mapping for PAQR8 antibodies?

Epitope mapping is crucial for understanding antibody specificity and can be performed through several approaches:

• Alanine scanning mutagenesis: Systematically substitute amino acids in the target region with alanine to identify critical binding residues, similar to the approach used for C8Mab-2 antibody against CCR8 where researchers identified the 17-DFFTAP-22 sequence as important for recognition

• Peptide array analysis: Test antibody binding against overlapping peptides spanning the target region (e.g., the C-terminal region 326-354 of PAQR8)

• Truncation analysis: Create truncated versions of the protein to narrow down the epitope region

• Computational prediction followed by experimental validation: Use bioinformatics tools to predict potential epitopes before experimental confirmation

• X-ray crystallography or cryo-EM: For detailed structural analysis of antibody-antigen complexes in advanced research settings

The choice of method depends on research goals, available resources, and required resolution of epitope information .

What approaches can be used to improve the specificity of PAQR8 antibodies?

Recent advances in antibody engineering provide several strategies to enhance specificity:

• Rational design approaches: Using complementary peptides designed specifically against target epitopes

• Biophysics-informed modeling: Combining computational design with experimental validation to optimize binding properties

• Selection against multiple similar targets: Including related proteins during negative selection to eliminate cross-reactive antibodies

• Identification of distinct binding modes: Using computational models to identify antibodies with unique binding signatures to specific epitopes

• Affinity maturation: Further optimization of binding properties through directed evolution approaches

These approaches can be applied to develop more specific PAQR8 antibodies, particularly for distinguishing between closely related PAQR family members.

How can researchers assess cross-reactivity of PAQR8 antibodies with other PAQR family members?

A systematic cross-reactivity assessment should include:

• Sequence alignment analysis: Identifying regions of homology between PAQR family members, particularly in the C-terminal epitope region (326-354 aa for the described antibody)

• Recombinant protein panel testing: Evaluating antibody binding against recombinant proteins of all PAQR family members

• Cell line panel analysis: Testing antibody specificity in cell lines with differential expression of PAQR family members

• Competitive binding assays: Using purified PAQR proteins to compete for antibody binding

• Knockout/knockdown validation: Confirming signal reduction in PAQR8-depleted samples while testing for unchanged signals in samples depleted of other PAQR family members

This comprehensive approach ensures the antibody is truly specific to PAQR8 rather than detecting related family members.

What are the best practices for using PAQR8 antibodies in Western blotting?

For optimal Western blotting results with PAQR8 antibodies:

• Sample preparation:

  • For membrane proteins like PAQR8, use appropriate detergent-based lysis buffers

  • Consider membrane fraction enrichment techniques

  • Avoid excessive heating which may cause membrane protein aggregation

• Gel selection and transfer:

  • Use gradient gels (4-12%) for better resolution

  • Consider specialized transfer conditions for membrane proteins (longer transfer times, lower voltage)

• Blocking and antibody incubation:

  • The recommended dilution for PAQR8 antibody is 1:1000

  • Optimize blocking conditions to minimize background

  • Consider longer incubation times at 4°C to improve signal-to-noise ratio

• Detection and analysis:

  • Include molecular weight markers to confirm the expected size (~40.5 kDa)

  • Use positive controls with known PAQR8 expression

  • Validate specificity with knockout/knockdown samples

These recommendations integrate general best practices for membrane protein Western blotting with specific information about the PAQR8 antibody.

How can researchers determine the optimal antibody concentration for their specific experimental conditions?

A systematic titration approach includes:

• Initial broad-range titration: Test several dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000) using positive control samples

• Signal-to-noise optimization: Calculate signal-to-background ratios for each concentration

• Fine-tuning: Perform narrower dilution ranges around the best-performing concentration

• Application-specific considerations:

  • For Western blotting, the recommended starting dilution is 1:1000

  • For IHC/IF, typically start with higher concentrations than WB

  • For flow cytometry, consider manufacturer recommendations for similar antibodies

• Sample-specific adjustment: Further optimize based on PAQR8 expression levels in experimental samples

This methodology ensures the most efficient use of antibody while maximizing specific signal detection.

What strategies can be used to distinguish between specific and non-specific binding in PAQR8 antibody experiments?

Researchers should implement multiple validation strategies:

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide (amino acids 326-354 of PAQR8) should abolish specific signals

• PAQR8 knockdown/knockout validation: Compare signals between wild-type and PAQR8-depleted samples

• Multiple antibody validation: Use antibodies targeting different epitopes of PAQR8 to confirm signals

• Cross-species validation: Test antibody in species where it is expected to work based on epitope conservation

• Signal correlation with known biology: Confirm that signals appear in tissues/conditions where PAQR8 is expected to be expressed

These approaches collectively provide strong evidence for binding specificity and follow established methodological principles in antibody research.

What are common issues when working with antibodies against membrane proteins like PAQR8?

These troubleshooting approaches address challenges specific to membrane proteins while following methodological principles applicable to antibody-based experiments in general.

How can researchers develop custom PAQR8 antibodies for specific research applications?

For developing specialized PAQR8 antibodies:

• Epitope selection:

  • Choose unique regions with low homology to other PAQR family members

  • Consider accessibility in the native protein conformation

  • For membrane proteins like PAQR8, extracellular loops often make good targets

• Immunization strategy:

  • Use KLH-conjugated synthetic peptides similar to the approach used for the commercial PAQR8 antibody

  • Consider DNA immunization for conformational epitopes

  • Implement negative selection against related proteins

• Screening methodology:

  • Employ Cell-Based Immunization and Screening methods similar to those used for CCR8 antibodies

  • Include counter-screening against related PAQR family members

  • Test for functionality in intended applications

• Rational design approaches:

  • Consider computational methods to design antibodies with customized specificity profiles

  • Implement biophysics-informed modeling for optimizing binding properties

• Production and purification:

  • For polyclonal antibodies, use affinity purification with the target peptide

  • For monoclonal antibodies, consider hybridoma technology or phage display

This comprehensive approach integrates methodological insights from various antibody development strategies in the current literature .

What are the key considerations when using PAQR8 antibodies for investigating protein-protein interactions?

When studying PAQR8 interactions, researchers should consider:

• Preservation of native interactions:

  • Use mild lysis conditions to maintain protein-protein interactions

  • Consider crosslinking approaches for transient interactions

  • Validate interactions under different detergent conditions

• Co-immunoprecipitation optimization:

  • Test different antibody immobilization strategies

  • Optimize antibody-to-lysate ratios

  • Consider using tagged PAQR8 constructs as complementary approaches

• Confirmation with multiple methods:

  • Validate interactions using reverse co-IP

  • Implement proximity ligation assays

  • Consider FRET/BRET approaches for live-cell interaction studies

• Controls for specificity:

  • Include isotype controls

  • Use PAQR8 knockout/knockdown samples

  • Compare with structurally related but functionally distinct PAQR family members

• Functional validation:

  • Confirm biological relevance of identified interactions

  • Investigate the impact of hormone binding on protein interactions

  • Map interaction domains through mutational analysis

These methodological considerations ensure robust investigation of PAQR8 interactions following established principles in protein interaction studies.