Recombinant Human GPRC6A Protein: Research Guide & Protocol

Recombinant Human GPRC6A Protein is a class C G protein-coupled receptor research reagent produced for use in biochemical, cell-based, and structural studies of nutrient sensing, metabolic regulation, and multi-ligand GPCR signaling.

Product Name

Recombinant Human GPRC6A Protein

SKU

rp368193

Protein Family

Class C G protein-coupled receptor (GPCR)

Species Origin

Human (Homo sapiens)

Primary Research Areas

Metabolic signaling, bone homeostasis, endocrine function, nutrient sensing

Recommended Storage

-80 °C for long-term storage; avoid repeated freeze-thaw cycles

Quality Reference

absea#PQ-407

Biological Background of GPRC6A

GPRC6A (G Protein-Coupled Receptor Class C Group 6 Member A) is a member of the class C family of GPCRs, which is characterized by a large extracellular Venus flytrap (VFT) domain responsible for ligand binding. GPRC6A functions as a multi-ligand receptor capable of sensing basic amino acids (such as L-arginine and L-ornithine), divalent cations (including calcium and magnesium), and the bone-derived hormone osteocalcin.

GPRC6A signals primarily through the G_αs and G_αi pathways, modulating intracellular cAMP levels in a cell-type-dependent manner. The receptor is broadly expressed across tissues including liver, kidney, skeletal muscle, testes, and bone, consistent with its roles in energy metabolism, testosterone biosynthesis, insulin secretion, and bone homeostasis.

Dysregulation of GPRC6A has been associated with conditions such as type 2 diabetes, prostate cancer progression, and osteoporosis, making it a compelling target for both basic research and therapeutic development.

Protein Specifications and Quality Standards

Recombinant Human GPRC6A Protein (rp368193) is produced according to quality reference absea#PQ-407. As a recombinant GPCR, the protein is typically expressed using mammalian or insect cell expression systems to preserve native glycosylation patterns and tertiary structure critical for ligand-binding activity.

Researchers should consult the product datasheet for lot-specific information on the following parameters:

• Purity: Assessed by SDS-PAGE and/or HPLC; typical threshold ≥85% for functional studies (verify with lot certificate).
• Formulation: Check the Certificate of Analysis (CoA) for buffer composition, stabilizers, and protein concentration.
• Biological Activity: Confirm via functional assays such as cAMP accumulation, ligand-binding assays, or receptor internalization studies as specified in the product documentation.
• Endotoxin Level: Verify endotoxin limits on the CoA, particularly for cell-based assays where endotoxin interference is a concern.

All specifications should be confirmed against the current product datasheet and CoA provided with each lot, as parameters may be updated to reflect manufacturing improvements.

Handling, Reconstitution, and Storage Protocol

Proper handling of Recombinant Human GPRC6A Protein is essential to maintain activity and reproducibility. Follow the steps below and always refer to the specific product datasheet for any lot-specific instructions that supersede general guidelines.

Upon Receipt

• Inspect the package for signs of temperature excursion during shipping.
• Record the lot number and expiration date in your laboratory inventory system.
• If the product arrives lyophilized, proceed with reconstitution as described below. If supplied in liquid form, aliquot immediately to avoid freeze-thaw cycles.

Reconstitution (Lyophilized Format)

1. Briefly centrifuge the vial (1,000 × g, 30 seconds) to collect material at the bottom before opening.
2. Reconstitute with the volume of sterile, ultrapure water or buffer recommended in the datasheet, typically to yield a stock concentration of 0.1–1.0 mg/mL.
3. Gently pipette to mix; avoid vortexing to prevent denaturation of the GPCR.
4. Allow to stand at room temperature for 5–10 minutes to ensure complete dissolution.

Aliquoting and Storage

• Prepare single-use aliquots to minimize freeze-thaw cycles, which can reduce receptor integrity and activity.
• Store aliquots at −80 °C for long-term stability.
• For short-term use (up to 1 week), store at 4 °C; do not refreeze after thawing.
• Add a carrier protein (e.g., 0.1% BSA) if diluting to very low concentrations to prevent adsorption to vessel surfaces.

Research Applications

Recombinant Human GPRC6A Protein supports a wide range of experimental applications in basic and translational research:

• Ligand Binding Assays: Characterize the binding kinetics and affinities of amino acids, osteocalcin, and cationic ligands to the GPRC6A VFT domain using fluorescence polarization, SPR, or radioligand displacement assays.
• Functional Signaling Studies: Use reconstitution systems or cell-free assays to study G-protein coupling efficiency (G_αs/G_αi) and downstream cAMP modulation.
• Structural Biology: Suitable as antigen for antibody generation or as a component in cryo-EM and X-ray crystallography studies aimed at resolving GPRC6A's receptor architecture.
• Drug Discovery and Screening: Employ as a target in high-throughput screening (HTS) campaigns to identify agonists, antagonists, or allosteric modulators with therapeutic relevance to metabolic disease, prostate cancer, or osteoporosis.
• Immunoassay Development: Serve as a standard or coating antigen in ELISA, Western blot validation, or immunoprecipitation experiments.
• Metabolic and Endocrine Research: Investigate GPRC6A's role in insulin secretion, testosterone production, and osteocalcin signaling pathways.

Troubleshooting Common Issues

The following guidance addresses common challenges encountered when working with Recombinant Human GPRC6A Protein:

Issue	Likely Cause	Recommended Action
Loss of activity after thawing	Freeze-thaw damage to receptor conformation	Pre-aliquot before first freeze; add 0.1% BSA as stabilizer; avoid repeated freeze-thaw cycles
Aggregation or precipitation	Improper reconstitution or incompatible buffer	Reconstitute gently without vortexing; confirm buffer compatibility with datasheet; centrifuge at 10,000 × g for 5 min to remove aggregates before use
Low signal in binding assay	Protein adsorption to assay vessel or suboptimal ligand concentration	Block assay vessels with BSA or other carrier; optimize ligand concentration range; verify protein concentration by BCA or Bradford assay
High background in cell-based assays	Endotoxin contamination	Verify endotoxin level on CoA; use LAL assay to test working stock; if elevated, use endotoxin-removal kit
No detectable cAMP response	Incorrect G-protein coupling conditions or receptor misfolding	Optimize reconstitution conditions; ensure appropriate G-protein is present; confirm receptor integrity by SDS-PAGE or mass spectrometry

Frequently asked questions

What is GPRC6A and why is it an important research target?

GPRC6A is a class C G protein-coupled receptor that acts as a multi-ligand sensor for amino acids, divalent cations, and the hormone osteocalcin. It plays roles in metabolic regulation, testosterone biosynthesis, insulin secretion, and bone homeostasis. Its association with metabolic disorders, prostate cancer, and osteoporosis makes it a compelling target for both basic mechanistic studies and therapeutic discovery.

How should Recombinant Human GPRC6A Protein be stored to maintain activity?

For long-term storage, keep the protein at −80 °C in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise receptor conformation and activity. For short-term use, the protein may be stored at 4 °C for up to one week. Always consult the lot-specific CoA for any storage conditions that differ from general guidelines.

Which expression system is used to produce this recombinant GPRC6A protein?

Class C GPCRs like GPRC6A typically require eukaryotic expression systems—most commonly mammalian (e.g., HEK293) or insect cell (e.g., Sf9 via baculovirus) platforms—to ensure proper folding, post-translational modifications, and functional activity. Please refer to the product datasheet for rp368193 for the specific expression system used for this lot.

Can Recombinant Human GPRC6A Protein be used for antibody production?

Yes, recombinant GPRC6A protein is suitable for use as an immunogen for generating GPRC6A-specific antibodies in animal immunization programs, and as a positive control antigen for validating anti-GPRC6A antibodies in Western blot, ELISA, and immunoprecipitation experiments. Confirm protein purity and integrity before immunization using SDS-PAGE.

What assay formats are compatible with Recombinant Human GPRC6A Protein?

Recombinant Human GPRC6A Protein is compatible with multiple assay formats including ligand-binding assays (fluorescence polarization, SPR, radioligand displacement), cAMP-based functional assays, ELISA (as coating antigen or standard), Western blot, immunoprecipitation, and structural studies (cryo-EM, X-ray crystallography). Optimize protein concentration and buffer conditions for each specific application.

References

1. Wellendorph P, Bräuner-Osborne H. Molecular cloning, expression, and sequence analysis of GPRC6A, a novel family C G-protein-coupled receptor. Gene. 2004;335:37-46. doi:10.1016/j.gene.2004.03.003
2. Pi M, Quarles LD. GPRC6A mediates multiple endocrine functions. Molecular and Cellular Endocrinology. 2012;353(1-2):69-74. doi:10.1016/j.mce.2011.11.019
3. Oury F, Sumara G, Sumara O, et al. Endocrine regulation of male fertility by the skeleton. Cell. 2011;144(5):796-809. doi:10.1016/j.cell.2011.02.004
4. Abreu AP, Noel SD, Xu S, et al. Evidence of the importance of the amino acid sensing receptor GPRC6A for metabolic and reproductive function. Molecular and Cellular Endocrinology. 2020;515:110924. doi:10.1016/j.mce.2020.110924

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.