paqr5b Antibody

What is PAQR5B and why is it important in research?

PAQR5B is a membrane progesterone receptor γ (mPRγ) that has been identified as essential for neuronal formation, particularly in the olfactory receptor (OR) of zebrafish. Research has demonstrated that Paqr5b-deficient zebrafish lack neurons in their olfactory epithelia, making it the first reported membrane progesterone receptor with a distinct role in neuronal development . The human homolog PAQR5 has been implicated in kidney cancer research, with low expression correlating with poor prognosis in kidney clear cell carcinoma (KIRC) . The protein's established role in neuronal development and potential as a prognostic marker in cancer makes it an important target for diverse research applications.

What tissue types express PAQR5B protein?

PAQR5B protein expression has been documented in several tissue types:

• Neural tissues: In zebrafish, Paqr5b is predominantly expressed in olfactory sensory neurons (OSNs), including ciliated OSNs with long dendrites, microvillus neurons with short dendrites, and crypt neurons in the apical region of the olfactory receptor .

• Kidney tissues: PAQR5 expression has been detected in normal kidney tissues, with significantly reduced expression in kidney clear cell carcinoma .

• Other mammalian tissues: Commercial antibodies for PAQR5/MPRG have been developed with confirmed reactivity to human, mouse, and rat samples, indicating conserved expression across these species .

Immunohistochemical analyses using specific antibodies have been essential for mapping the tissue distribution of this protein.

How can I validate the specificity of a PAQR5B antibody?

Validating PAQR5B antibody specificity requires multiple complementary approaches:

• Genetic validation: Compare staining between wild-type and knockout/knockdown models. Zebrafish studies effectively demonstrated antibody specificity by comparing paqr5b+/+ and paqr5b-/- samples, showing complete absence of the Paqr5b protein band in knockout samples .

• Recombinant protein testing: Confirm antibody reactivity with recombinant Paqr5b protein expressed in systems like yeast .

• Western blot analysis: Verify that the observed molecular weight matches expectations (approximately 55 kDa for zebrafish Paqr5b, which appears as a glycoprotein, or 72 kDa for human PAQR5) .

• Immunohistochemical controls: Perform parallel staining on tissues known to be positive or negative for expression, with careful attention to control tissues processed identically except for primary antibody omission.

• Peptide competition: Pre-adsorb the antibody with the immunizing peptide to confirm binding specificity.

These validation steps are crucial for ensuring reliable research outcomes and should be documented thoroughly in publications.

What are the typical molecular weights observed for PAQR5B in Western blot analysis?

Western blot analysis of PAQR5B reveals interesting differences between observed and predicted molecular weights:

• Zebrafish Paqr5b: Detected as a broad protein band with a molecular weight of approximately 55 kDa, which is presumed to be glycosylated .

• Human PAQR5: Commercial antibodies report an observed molecular weight of 72 kDa, while the calculated molecular weight based on amino acid sequence is approximately 38 kDa (38,014 Da) .

The significant discrepancy between observed and calculated molecular weights is likely due to post-translational modifications, particularly glycosylation. When troubleshooting Western blots, researchers should be aware that membrane proteins like PAQR5B often migrate at higher apparent molecular weights than predicted. Using appropriate positive controls and molecular weight markers is essential for accurate band identification.

What immunohistochemical protocols have been successful for PAQR5B detection?

Successful immunohistochemical detection of PAQR5B has been achieved using the following protocols:

For paraffin-embedded tissues:

• Section thickness: 3-μm

• Primary antibody: Anti-PAQR5 antibody (such as ab236798, Abcam)

• Dilution ratio: 1/200

• Incubation conditions: 2 hours at 37°C

• Secondary detection: Horseradish peroxidase antibody (1 hour at room temperature)

• Visualization: 3,3-diaminobenzidine (DAB) for 5 minutes

• Counterstaining: Hematoxylin for 1 minute

For zebrafish tissues specifically, immunofluorescence protocols utilizing anti-Paqr5b antibodies have successfully demonstrated co-localization with neuronal markers like acetylated tubulin . Both DAB-based and fluorescence-based detection methods have proven effective, with choice depending on research needs (quantification vs. co-localization studies).

How does PAQR5B expression differ between normal and pathological tissues?

Research has revealed significant differences in PAQR5B expression between normal and pathological states:

These differential expression patterns highlight PAQR5B's potential as both a diagnostic biomarker and a therapeutic target, particularly in oncology research.

What are the most effective methods for studying PAQR5B's role in neuronal development?

To investigate PAQR5B's role in neuronal development, researchers have successfully employed several complementary approaches:

• Genetic manipulation:

  • CRISPR/Cas9-mediated gene editing to generate knockout models, as demonstrated in zebrafish paqr5b studies

  • Heteroduplex mobility analysis (HMA) and DNA sequencing to confirm genetic modifications

  • Crossing of heterozygous models to obtain homozygous mutants for comparative studies

• Protein expression analysis:

  • Western blot analysis using specific anti-Paqr5b antibodies

  • Immunohistochemistry to localize expression in specific cell types

  • Co-immunostaining with neuronal markers (such as acetylated tubulin) to confirm neuronal expression

• Histological assessment:

  • H&E staining to evaluate tissue architecture and cell populations

  • Alcian blue staining to identify mucoid components that might interfere with antibody specificity

• Functional studies:

  • Cell proliferation markers (phosphorylated histone H3)

  • Apoptosis markers (cleaved-caspase3)

  • Behavioral assays to assess functional consequences of PAQR5B deficiency

These methodologies provide a comprehensive framework for investigating PAQR5B's neurobiological functions.

How can I design experiments to correlate PAQR5B expression with cancer progression?

Based on kidney cancer research findings, the following experimental design would effectively correlate PAQR5B expression with cancer progression:

• Sample collection and characterization:

  • Paired tumor and adjacent normal tissues from patients with well-documented clinical characteristics

  • Comprehensive documentation of tumor stage, grade, and nodal metastasis status

  • Patient follow-up data for survival analysis

• Expression analysis:

  • Immunohistochemical staining with validated anti-PAQR5 antibodies

  • Quantitative image analysis to measure staining intensity and distribution

  • Western blot quantification with appropriate loading controls

  • Correlation with mRNA expression data from the same samples

• Statistical approaches:

  • ROC curve analysis to assess diagnostic potential

  • Kaplan-Meier survival analysis stratified by PAQR5 expression levels

  • Cox regression analysis to identify independent prognostic factors

  • Correlation analysis with established cancer markers

• Mechanistic investigations:

  • Gene Ontology term analysis and pathway analysis (KEGG)

  • Gene set enrichment analysis (GSEA)

  • Protein-protein interaction mapping (using tools like STRING and GeneMANIA)

  • Correlation with immune cell infiltration and immunomodulatory molecules

This comprehensive approach enables both clinical correlation and mechanistic insights into PAQR5B's role in cancer biology.

What signaling pathways interact with PAQR5B and how can these be studied?

Research has identified several signaling pathways that interact with PAQR5B, providing opportunities for mechanistic studies:

• Identified pathway interactions:

  • STAT signaling pathway: PAQR5 expression negatively correlates with STAT1/2/3/4/5A in kidney cancer

  • HIF-1α pathway: Negative correlation observed in KIRC

  • mTOR signaling: Negative correlation in KIRC

  • Immune checkpoint molecules: Negative correlation with PD-1, CTLA4, and LAG3

• Experimental approaches to study these interactions:

  • Co-immunoprecipitation to detect physical interactions

  • Proximity ligation assays for in situ detection of protein-protein interactions

  • Pathway inhibitor studies to assess functional relationships

  • Gene expression analysis after PAQR5B manipulation

• Functional readouts:

  • Phosphorylation status of pathway components

  • Transcriptional activity of downstream targets

  • Cellular phenotypes (proliferation, differentiation, migration)

  • Immune cell infiltration and activation states

Understanding these pathway interactions provides insights into both physiological functions and pathological alterations of PAQR5B signaling.

How can I distinguish between PAQR5B's genomic and non-genomic actions in experimental models?

As a membrane progesterone receptor, PAQR5B may exhibit both genomic and non-genomic effects. Distinguishing between these mechanisms requires specific experimental approaches:

• Temporal analysis:

  • Non-genomic effects typically occur rapidly (seconds to minutes)

  • Genomic effects generally require hours for transcription and translation

  • Time-course experiments can separate these responses

• Subcellular localization studies:

  • Membrane-localized PAQR5B mediates non-genomic effects

  • Co-localization with signaling components like G-proteins suggests non-genomic pathways

  • Translocation to the nucleus would suggest potential genomic regulation

• Signaling pathway analysis:

  • Second messenger activation (cAMP, calcium) indicates non-genomic signaling

  • Transcription factor activation suggests genomic regulation

  • Phosphorylation cascades can contribute to both pathways

• Inhibitor studies:

  • Transcription or translation inhibitors will block genomic but not non-genomic effects

  • Membrane-impermeable conjugates of progesterone can isolate membrane-initiated signaling

  • G-protein inhibitors can disrupt non-genomic pathways

These experimental strategies can provide mechanistic insights into the diverse functions of PAQR5B in different cellular contexts.

What are the critical control experiments when using PAQR5B antibodies?

Robust experimental design requires comprehensive controls when working with PAQR5B antibodies:

• Negative controls:

  • Omission of primary antibody (secondary antibody only)

  • Isotype control antibody (same species and isotype as anti-PAQR5B)

  • Tissues known not to express PAQR5B

  • Knockout/knockdown samples when available (like paqr5b-/- zebrafish)

  • Pre-adsorption with immunizing peptide

• Positive controls:

  • Tissues with confirmed PAQR5B expression (olfactory sensory neurons for zebrafish)

  • Recombinant PAQR5B protein for Western blot

  • Previously validated positive samples

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Processing controls to ensure consistent immunostaining

  • Cross-reactivity controls when using multiple antibodies

Including appropriate controls not only validates results but also helps troubleshoot technical issues. The zebrafish knockout model has proven particularly valuable as a negative control for antibody specificity .

How can I optimize Western blot protocols specifically for PAQR5B detection?

Western blot optimization for PAQR5B detection requires attention to several critical factors:

• Sample preparation:

  • For zebrafish olfactory receptors, direct homogenization in SDS sample buffer has been effective

  • Include protease inhibitors to prevent degradation

  • Process samples at 4°C to maintain protein integrity

• Gel electrophoresis and transfer:

  • 12% SDS-PAGE gels provide good resolution for PAQR5B

  • Transfer to PVDF membranes (0.45 μm pore size) at 15V and 400 mA for 45 minutes using a semi-dry system

• Blocking and antibody incubation:

  • Block with 5% skim milk powder in TBS containing 0.1% Tween-20 (TTBS) at 4°C overnight

  • Incubate with primary antibody (1:1000 dilution) for 2 hours at 4°C

  • Wash thoroughly with TTBS (3 times, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:2000) for 2 hours at 4°C

• Detection and analysis:

  • Use enhanced chemiluminescence detection systems

  • Capture images with appropriate exposure to avoid saturation

  • Analyze with quantitative software like ImageQuant TL

This optimized protocol has successfully detected both endogenous Paqr5b from zebrafish tissues and recombinant PAQR5B proteins.

What are the key considerations for immunohistochemical localization of PAQR5B?

Successful immunohistochemical localization of PAQR5B requires attention to several technical factors:

• Tissue processing:

  • Optimal fixation is critical - overfixation can mask epitopes while underfixation compromises tissue morphology

  • 3-μm section thickness has been effective for PAQR5 detection in paraffin-embedded tissues

  • Consistent processing across all experimental groups is essential for comparative studies

• Antigen retrieval:

  • Heat-induced epitope retrieval may be necessary for formalin-fixed tissues

  • Optimization of retrieval conditions (pH, buffer composition, time) is recommended

• Antibody selection and optimization:

  • Validate antibody specificity with appropriate controls

  • Titrate antibody concentration (1:200 dilution has been effective for human tissues)

  • Optimize incubation conditions (2 hours at 37°C has been reported as effective)

• Signal detection and analysis:

  • Both chromogenic (DAB) and fluorescent detection systems can be used

  • For co-localization studies, immunofluorescence with confocal microscopy provides superior results

  • Digital image analysis enables quantitative assessment of expression patterns

• Special considerations:

  • Be aware that mucoid coverings of tissues like olfactory epithelium may bind antibodies non-specifically, as demonstrated with Alcian blue staining

  • Co-staining with cell type-specific markers can help identify expressing populations

These optimized approaches have successfully demonstrated PAQR5B expression in both neuronal and kidney tissues.

How can I quantitatively analyze PAQR5B expression data from immunostaining experiments?

Quantitative analysis of PAQR5B immunostaining requires systematic approaches:

• Image acquisition considerations:

  • Consistent microscope settings across all samples

  • Representative sampling of tissue areas

  • Sufficient resolution to detect cellular and subcellular features

  • Inclusion of reference standards for intensity calibration

• Analytical methods:

  • H-score methodology (combines intensity and percentage of positive cells)

  • Automated image analysis using specialized software

  • Cell counting for specific populations (e.g., neurons in the olfactory epithelium)

  • Intensity measurement normalized to background

• Statistical approaches:

  • Appropriate normalization to control for technical variation

  • Non-parametric tests for intensity data (often not normally distributed)

  • Analysis of variance (ANOVA) for multiple group comparisons

  • Correlation analysis with clinical parameters or experimental variables

• Presentation formats:

  • Box plots or violin plots for distribution visualization

  • Representative images alongside quantitative data

  • Clear indication of statistical tests and significance levels

For kidney cancer research, comparing PAQR5 expression between tumor and normal tissues using these quantitative approaches has demonstrated significant diagnostic and prognostic value (AUC = 0.962) .

What protocols are recommended for co-immunoprecipitation studies targeting PAQR5B?

Co-immunoprecipitation (Co-IP) of PAQR5B requires special considerations for membrane protein isolation and interaction preservation:

• Sample preparation:

  • Gentle lysis buffers containing mild detergents (CHAPS, digitonin, or NP-40)

  • Inclusion of protease and phosphatase inhibitors

  • Maintaining physiological pH and salt concentrations

  • Pre-clearing lysates to reduce non-specific binding

• Antibody selection:

  • Use antibodies validated for immunoprecipitation applications

  • Consider epitope location relative to interaction domains

  • For zebrafish studies, custom antibodies against Paqr5b have been developed

  • For human studies, commercial antibodies with validated IP applications should be selected

• IP procedure:

  • Pre-couple antibodies to protein A/G beads or magnetic beads

  • Optimize antibody:lysate ratios

  • Include appropriate controls (IgG control, input fraction)

  • Gentle washing to preserve interactions

• Detection methods:

  • Western blot analysis of precipitated complexes

  • Mass spectrometry for unbiased identification of interaction partners

  • Reciprocal Co-IP to confirm interactions

Based on pathway correlations identified in kidney cancer research, potential interaction partners to investigate include STAT pathway proteins, HIF-1α, and mTOR pathway components .

How should I interpret differences between mRNA and protein expression levels of PAQR5B?

Discrepancies between PAQR5B mRNA and protein levels can provide important biological insights:

• Observed patterns in research:

  • In zebrafish paqr5b-/- studies, qPCR analysis revealed that mRNA expression levels were not different from wild-type, despite the complete absence of protein in Western blot analysis

  • This suggests significant post-transcriptional regulation

• Potential mechanisms explaining discrepancies:

  • Post-transcriptional regulation (miRNA-mediated repression, RNA-binding proteins)

  • Nonsense-mediated decay for mutant transcripts

  • Differences in half-life between mRNA and protein

  • Translational efficiency variations

  • Post-translational modifications affecting antibody recognition

• Analytical approaches:

  • Compare mRNA quantification (qPCR) with protein levels (Western blot)

  • Assess temporal relationships between transcript and protein expression

  • Investigate potential regulatory mechanisms (miRNA profiling, polysome analysis)

  • Consider technical factors (primer design, antibody specificity)

These observations highlight the importance of assessing both mRNA and protein levels when studying PAQR5B, particularly in genetic manipulation studies or disease models.

What are the most common technical challenges when working with PAQR5B antibodies?

Researchers frequently encounter several technical challenges when working with PAQR5B antibodies:

• Specificity issues:

  • Cross-reactivity with related PAQR family members

  • Non-specific binding to glycan-rich structures like mucoid coverings in olfactory epithelia

  • Variability between antibody lots

  • Species cross-reactivity limitations

• Detection challenges:

  • Post-translational modifications affecting epitope recognition

  • Membrane protein solubilization difficulties

  • Discrepancy between predicted and observed molecular weights due to glycosylation

  • Signal strength variability across different applications

• Application-specific issues:

  • For IHC: Antigen masking during fixation

  • For Western blot: Complete transfer of membrane proteins

  • For immunofluorescence: Autofluorescence in certain tissues

  • For flow cytometry: Maintaining protein conformation during cell preparation

• Troubleshooting approaches:

  • Comparison across multiple antibodies targeting different epitopes

  • Optimization of sample preparation (membrane protein extraction protocols)

  • Inclusion of appropriate positive and negative controls

  • Deglycosylation treatments to confirm protein identity

The use of knockout models, like the paqr5b-/- zebrafish, provides definitive negative controls that can help address many of these challenges .

How do I reconcile contradictory findings about PAQR5B expression across different studies?

When facing contradictory findings about PAQR5B expression across studies, consider these analytical approaches:

• Methodological differences:

  • Antibody selection (epitope location, clone, species reactivity)

  • Detection methods (IHC vs. Western blot vs. RT-PCR)

  • Sample preparation protocols

  • Quantification approaches

• Biological variables:

  • Species differences (zebrafish vs. human vs. rodent models)

  • Tissue-specific expression patterns

  • Developmental stage variations

  • Disease state or experimental condition influences

• Technical considerations:

  • Specificity validation methods used

  • Control samples included

  • Statistical approaches applied

  • Definition of "positive" expression

• Resolution strategies:

  • Direct comparison using standardized protocols

  • Meta-analysis of available data

  • Independent validation with orthogonal methods

  • Consideration of biological context for each study

The varying molecular weights reported for PAQR5B (55 kDa in zebrafish vs. 72 kDa in human studies ) illustrate how species differences and post-translational modifications can contribute to apparently contradictory findings that actually reflect biological variation.

What experimental approaches can distinguish between specific PAQR5B isoforms?

Distinguishing between potential PAQR5B isoforms requires specialized experimental approaches:

• Molecular characterization:

  • RT-PCR with isoform-specific primers spanning relevant exon junctions

  • Northern blot analysis to identify transcript size variations

  • Targeted RNA sequencing to identify alternative splicing events

  • 5' and 3' RACE to characterize transcript ends

• Protein-level discrimination:

  • Western blot analysis with antibodies targeting isoform-specific regions

  • 2D gel electrophoresis to separate based on both size and charge

  • Mass spectrometry for precise identification of protein variants

  • Isoform-specific immunoprecipitation

• Functional assessment:

  • Isoform-specific knockdown/knockout studies

  • Heterologous expression of individual isoforms

  • Domain-specific mutational analysis

  • Subcellular localization studies

• Bioinformatic approaches:

  • Sequence alignment to identify conserved and variable regions

  • Structural prediction to assess functional implications of isoform differences

  • Expression correlation analysis across tissues and conditions

These approaches can help researchers determine whether observed variations in PAQR5B expression or function result from distinct isoforms or post-translational modifications.

How can I design experiments to elucidate PAQR5B's role in disease progression?

Based on current knowledge of PAQR5B's implications in disease, particularly in kidney cancer, the following experimental design would effectively investigate its role in disease progression:

• Expression profiling across disease stages:

  • Comprehensive immunohistochemical analysis across disease progression stages

  • Correlation with established disease markers

  • Longitudinal sampling when possible

  • Integration with clinical outcome data

• Mechanistic investigations:

  • Genetic manipulation of PAQR5B in relevant cell lines or animal models

  • Assessment of phenotypic changes (proliferation, migration, invasion)

  • Pathway analysis focusing on identified correlations (STAT signaling, HIF-1α, mTOR)

  • Immune infiltration analysis (given PAQR5's correlation with immune cell populations)

• Therapeutic implications:

  • Restoration of PAQR5B expression in deficient models

  • Identification of compounds that modulate PAQR5B expression or function

  • Combination studies with established treatments

  • Biomarker development for patient stratification

• Clinical translation:

  • Development of standardized assays for PAQR5B assessment

  • Correlation with treatment response

  • Integration into prognostic models

  • Prospective validation in clinical cohorts

This comprehensive approach would build upon the established correlations between PAQR5B expression and disease outcomes, particularly the finding that PAQR5B upregulation is an independent factor for good prognosis in kidney cancer .