paqr7b Antibody

What is PAQR7B and how does it relate to PAQR7?

PAQR7B is a paralog of the PAQR7 gene, which encodes the progestin and adipoQ receptor family member 7 protein. PAQR7 (also known as membrane progestin receptor alpha, mPRα, MPRA, PGLP, or mSR) is a membrane protein with seven transmembrane domains, structurally similar to G protein-coupled receptors . PAQR7 has a molecular weight of approximately 39.7-40 kDa . While PAQR7B shares sequence homology with PAQR7, researchers should be aware of the specific differences when selecting antibodies for experimental applications.

What are the primary research applications for PAQR7B antibodies?

PAQR7B antibodies are valuable tools for multiple research applications, particularly in reproductive biology and endocrinology research. Based on the applications validated for related PAQR7 antibodies, common techniques include Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF), Immunohistochemistry (IHC), and Immunocytochemistry (ICC) . The selection of appropriate application should be guided by specific experimental objectives and validated reactivity patterns.

How do I determine the appropriate PAQR7B antibody for my research?

When selecting a PAQR7B antibody, researchers should consider several critical factors:

• Species reactivity: Ensure the antibody recognizes PAQR7B in your experimental species (human, mouse, rat, etc.)

• Application validation: Verify the antibody has been validated for your specific application (WB, ELISA, IF, IHC)

• Epitope specificity: Consider whether the antibody targets a specific region (e.g., C-terminal) that is conserved in PAQR7B

• Clonality: Determine whether a polyclonal or monoclonal antibody is more appropriate for your experiment

• Host species: Select an antibody raised in a species compatible with your experimental design

What are the recommended protocols for Western Blot analysis using PAQR7B antibodies?

For effective Western Blot analysis with PAQR7B antibodies, researchers should consider the following protocol guidelines:

• Sample preparation: Extract proteins using appropriate lysis buffers that preserve membrane proteins

• Protein separation: Use SDS-PAGE gels (10-12%) for optimal resolution around the 40 kDa range

• Transfer conditions: Employ wet transfer methods for better transfer of membrane proteins

• Antibody dilution: For PAQR7-related antibodies, a dilution range of 1:500-1:3000 has been recommended for Western Blot applications

• Detection: Use chemiluminescence or fluorescence-based detection systems depending on sensitivity requirements

Human cell lines such as HEK-293, HeLa, and SW480 have been successfully used for PAQR7 detection and may serve as positive controls for PAQR7B studies .

How should immunohistochemistry experiments with PAQR7B antibodies be optimized?

Optimizing immunohistochemistry protocols for PAQR7B antibodies requires careful consideration of several factors:

• Fixation method: Choose appropriate fixatives (paraformaldehyde or formalin) that preserve epitope structure

• Antigen retrieval: Determine the optimal antigen retrieval method (heat-induced or enzymatic) through pilot experiments

• Blocking conditions: Use species-appropriate blocking sera to minimize background

• Antibody concentration: Titrate antibody concentrations to achieve optimal signal-to-noise ratio

• Incubation conditions: Optimize primary antibody incubation time and temperature

• Controls: Include both positive and negative controls to validate staining specificity

As PAQR7 has important physiological functions in various reproductive tissues , these tissues may serve as appropriate positive controls for PAQR7B studies.

What are the recommended storage conditions for maintaining PAQR7B antibody activity?

To preserve antibody functionality, researchers should follow these storage guidelines:

• Long-term storage: Store at -20°C for up to one year

• Working solutions: For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles, which can degrade antibody performance

• Consider aliquoting antibodies upon receipt to minimize freeze-thaw cycles

• Store in appropriate buffer conditions, typically PBS with glycerol (often 50%) and stabilizers like BSA (0.5-1%)

How can PAQR7B antibodies be employed in co-immunoprecipitation studies to investigate protein-protein interactions?

For co-immunoprecipitation (Co-IP) studies investigating PAQR7B interactions:

• Cross-linking: Consider using membrane-permeable cross-linkers to stabilize transient protein interactions

• Lysis conditions: Use mild, non-denaturing detergents (such as NP-40 or Digitonin) to preserve protein complexes

• Pre-clearing: Remove non-specific binding proteins with appropriate control IgG

• Antibody selection: Choose antibodies with validated specificity for IP applications

• Washing stringency: Optimize washing conditions to balance between removing non-specific interactions and preserving genuine interactions

• Controls: Include IgG controls and lysate-only controls to identify non-specific binding

This approach is particularly valuable for investigating PAQR7B's potential interactions with progesterone signaling pathway components, given PAQR7's established role in progesterone signaling .

What strategies can be employed to validate PAQR7B antibody specificity for research applications?

Rigorous validation of PAQR7B antibody specificity is essential and can be accomplished through:

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm specificity

• Genetic approaches: Use PAQR7B knockout or knockdown models as negative controls

• Recombinant protein controls: Test antibody against purified recombinant PAQR7B protein

• Orthogonal detection methods: Confirm findings using multiple antibodies targeting different epitopes

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins

• Cross-reactivity testing: Evaluate potential cross-reactivity with closely related proteins, particularly PAQR7

How can researchers distinguish between PAQR7 and PAQR7B in experimental systems?

Differentiating between these related proteins requires careful experimental design:

• Antibody selection: Use antibodies raised against unique, non-conserved epitopes specific to either PAQR7 or PAQR7B

• RNA-level analysis: Employ RT-PCR or RNA-Seq with primers specific to unique regions of each transcript

• Genetic manipulation: Use targeted knockdown or knockout approaches specific to each gene

• Expression patterns: Compare tissue-specific expression patterns, as they may differ between the two proteins

• Functional assays: Develop specific functional readouts based on known or predicted differences in activity

What are common issues encountered in Western Blot analysis with PAQR7B antibodies and their solutions?

Researchers frequently encounter these challenges when working with membrane proteins like PAQR7B:

How can I optimize immunofluorescence experiments with PAQR7B antibodies?

For successful immunofluorescence detection of PAQR7B:

• Fixation optimization: Test multiple fixatives to determine which best preserves antigen accessibility

• Permeabilization: Carefully optimize detergent concentration and exposure time, as excessive permeabilization can disrupt membrane proteins

• Blocking parameters: Use species-appropriate normal serum with additional blocking agents like BSA

• Antibody concentration: Perform titration experiments to determine optimal antibody dilutions

• Incubation conditions: Compare overnight incubation at 4°C versus room temperature incubation

• Detection system: Select appropriate fluorophore-conjugated secondary antibodies with minimal spectral overlap to other fluorescent markers

What controls should be included when performing PAQR7B immunohistochemistry?

Essential controls for rigorous PAQR7B immunohistochemistry experiments include:

• Positive tissue controls: Tissues known to express PAQR7B

• Negative tissue controls: Tissues known not to express PAQR7B

• Primary antibody omission: To assess non-specific binding of secondary antibody

• Isotype control: Primary antibody replaced with non-specific IgG from the same species

• Absorption control: Primary antibody pre-incubated with immunizing peptide

• Genetic controls: When available, tissues from knockout or knockdown models

How can PAQR7B antibodies be utilized in single-cell protein analysis techniques?

Emerging single-cell protein analysis methods with PAQR7B antibodies include:

• Mass cytometry (CyTOF): Conjugate PAQR7B antibodies to metal isotopes for high-parameter single-cell analysis

• Single-cell Western blotting: Adapt traditional Western blot protocols for microfluidic platforms

• Proximity ligation assay (PLA): Detect protein-protein interactions involving PAQR7B at single-molecule resolution

• Imaging mass cytometry: Combine antibody-based detection with spatial resolution in tissue sections

• Multiplex immunofluorescence: Develop compatible antibody panels for simultaneous detection of multiple proteins

These approaches enable researchers to investigate PAQR7B expression and localization with unprecedented resolution.

What are the considerations for developing multiplex assays that include PAQR7B detection?

When developing multiplex assays incorporating PAQR7B antibodies:

• Antibody compatibility: Select antibodies raised in different host species to avoid cross-reactivity

• Signal separation: Choose detection systems with minimal spectral overlap

• Sequential staining: Consider sequential rather than simultaneous antibody incubations

• Signal amplification: Implement tyramide signal amplification for weak signals

• Validation: Thoroughly validate multiplex results against single-stain controls

• Quantification: Develop appropriate image analysis workflows for accurate quantification

How can researchers investigate the role of PAQR7B in progesterone signaling pathways?

Given PAQR7's established role in progesterone signaling , researchers investigating PAQR7B's potential functions can:

• Co-localization studies: Use PAQR7B antibodies alongside markers for progesterone receptors to assess spatial relationships

• Functional assays: Measure progesterone-induced calcium mobilization or cAMP production in cells with manipulated PAQR7B expression

• Pharmacological approaches: Compare cellular responses to progesterone versus membrane-impermeable progesterone conjugates

• Protein-protein interaction studies: Investigate interactions between PAQR7B and known progesterone signaling components

• Phosphorylation analysis: Examine progesterone-induced phosphorylation events in the presence and absence of PAQR7B