Recombinant Human GPR50 Protein: Production & Usage Protocol

Recombinant Human GPR50 Protein is a research-grade recombinant protein derived from the human GPR50 gene, designed to support GPCR functional studies, circadian rhythm research, and metabolic signaling investigations in vitro.

Product Name

Recombinant Human GPR50 Protein

SKU

rp368435

Protein Target

GPR50 (G Protein-Coupled Receptor 50)

Species

Human (Homo sapiens)

Application

GPCR binding assays, functional studies, circadian and metabolic research

Storage Recommendation

Store at -80°C; avoid repeated freeze-thaw cycles

Background and Scientific Overview

GPR50 (G Protein-Coupled Receptor 50) is an orphan G protein-coupled receptor belonging to the melatonin receptor subfamily. It shares significant structural homology with melatonin receptors MT1 (MTNR1A) and MT2 (MTNR1B), yet does not bind melatonin itself. GPR50 is predominantly expressed in the hypothalamus and pituitary gland, and has been implicated in circadian rhythm regulation, energy homeostasis, and metabolic processes.

Research into GPR50 has highlighted its potential roles in body weight regulation, lipid metabolism, and psychiatric conditions including bipolar disorder. As an orphan receptor, identifying endogenous ligands and understanding downstream signaling pathways remain active areas of investigation. Recombinant Human GPR50 Protein (rp368435) provides a reliable tool for such studies, enabling researchers to conduct binding assays, protein interaction studies, and functional characterization in controlled in vitro settings.

Materials and Equipment Required

Before beginning, ensure the following materials and equipment are available:

• Recombinant Human GPR50 Protein (rp368435, Aladdin Scientific)
• Sterile, nuclease-free water or recommended reconstitution buffer (e.g., PBS, pH 7.4)
• Microcentrifuge tubes (1.5 mL, low-protein-binding recommended)
• Calibrated micropipettes and sterile tips
• Benchtop microcentrifuge
• Ice bucket and dry ice for cold-chain handling
• -80°C freezer for long-term storage
• Protein quantification reagents (e.g., BCA or Bradford assay kit)
• SDS-PAGE and western blot supplies for QC verification (optional)
• Appropriate assay buffers compatible with downstream application (e.g., binding assay buffer, ELISA reagents)

Step-by-Step Reconstitution and Handling Protocol

Follow these steps carefully to ensure protein integrity and reproducible results:

1. Thawing: Remove the vial from -80°C storage and allow it to thaw slowly on ice at 4°C. Do not thaw at room temperature or use a water bath, as rapid temperature changes may compromise protein structure.
2. Brief Centrifugation: Before opening, briefly centrifuge the vial (1,000–2,000 × g, 30 seconds) to collect any lyophilized material or liquid from the cap and walls.
3. Reconstitution: If supplied in lyophilized form, add the recommended volume of sterile reconstitution buffer (typically sterile PBS at pH 7.4, or as specified in the product datasheet) gently along the vial wall. Do not vortex; instead, gently swirl or pipette slowly to dissolve the protein completely.
4. Equilibration: Allow the reconstituted solution to stand on ice for 5–10 minutes to ensure complete dissolution.
5. Quantification: Determine protein concentration using a BCA or Bradford assay. Compare against the certificate of analysis (CoA) value to confirm recovery.
6. Aliquoting: Divide the reconstituted protein into single-use aliquots to minimize freeze-thaw cycles. Label each aliquot with date, concentration, and lot number.
7. Storage: Store aliquots at -80°C for long-term use. For short-term use within 1–2 weeks, aliquots may be stored at -20°C. Avoid storing at 4°C for extended periods.

Note: Refer to the product-specific datasheet (absea#PP-3311) for exact buffer compositions, reconstitution volumes, and concentration specifications, as these may vary by lot.

Quality Control and Verification

Quality control is essential to confirm protein identity, purity, and activity before downstream use. Recommended QC steps include:

• SDS-PAGE: Run the reconstituted protein on a reducing SDS-PAGE gel. A single predominant band at the expected molecular weight confirms purity. Consult the product datasheet for the expected molecular weight of GPR50 recombinant protein.
• Western Blot: Confirm protein identity using an anti-GPR50 antibody or an antibody recognizing the incorporated purification tag (e.g., His-tag or GST-tag, as applicable).
• Protein Concentration Verification: Compare measured concentration with CoA specifications to ensure reconstitution was successful.
• Functional Assessment (Optional): If conducting binding or activity assays, include a positive control with a validated GPR50-binding partner or reference compound to confirm protein functionality.

Document all QC results with the corresponding lot number and storage conditions for traceability and reproducibility reporting.

Recommended Applications and Assay Guidance

Recombinant Human GPR50 Protein (rp368435) is suitable for a range of in vitro research applications, including but not limited to:

• Ligand Binding Assays: Use GPR50 protein to screen candidate ligands or small molecules in competitive binding formats. Establish binding kinetics and affinity constants under optimized buffer conditions.
• Protein–Protein Interaction Studies: Employ co-immunoprecipitation (co-IP), pull-down assays, or surface plasmon resonance (SPR) to identify GPR50 interaction partners relevant to circadian or metabolic signaling networks.
• ELISA-Based Detection: Use as a coating antigen or detection standard in sandwich or indirect ELISA formats to develop or validate GPR50-specific immunoassays.
• Structural Studies: Serve as antigen for antibody generation or as a reference standard in structural biology investigations.
• Cell-Free Functional Assays: Incorporate into cell-free systems to study receptor conformation, post-translational modifications, or downstream effector interactions.

Optimize protein concentration, buffer conditions, temperature, and incubation times empirically for each specific application. Include appropriate negative and positive controls in all experiments.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GPR50 Protein?

The general recommendation is sterile PBS at pH 7.4; however, the exact reconstitution buffer, volume, and concentration should be confirmed against the product-specific datasheet (absea#PP-3311) and Certificate of Analysis supplied with each lot, as formulations may vary.

How should Recombinant Human GPR50 Protein be stored to maintain stability?

The protein should be stored at -80°C for long-term preservation. Avoid repeated freeze-thaw cycles by aliquoting the reconstituted protein into single-use volumes before storage. For short-term use within 1–2 weeks, storage at -20°C is acceptable.

What downstream applications is Recombinant Human GPR50 Protein validated for?

Recombinant Human GPR50 Protein is suitable for ligand binding assays, protein-protein interaction studies (co-IP, pull-down, SPR), ELISA-based immunoassays, antigen preparation for antibody generation, and cell-free functional studies. Researchers should optimize conditions empirically for each application and confirm validated uses in the product datasheet.

How does GPR50 differ from melatonin receptors MT1 and MT2?

GPR50 shares significant amino acid sequence homology with the melatonin receptors MT1 and MT2, belonging to the same receptor subfamily. However, unlike MT1 and MT2, GPR50 does not bind melatonin and currently has no confirmed endogenous ligand, classifying it as an orphan receptor. It has been associated with circadian rhythm regulation, energy balance, and metabolic signaling through distinct mechanisms.

How can I verify the identity and purity of the GPR50 protein after reconstitution?

Identity and purity can be confirmed by running the reconstituted protein on a reducing SDS-PAGE gel to check for a predominant band at the expected molecular weight. Western blot using an anti-GPR50 antibody or an anti-tag antibody (if a purification tag is present) can confirm protein identity. Protein concentration should also be verified by BCA or Bradford assay and compared to the CoA specifications.

References

1. Reppert SM, et al. Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proceedings of the National Academy of Sciences. 1995.
2. Levoye A, et al. The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization. EMBO Journal. 2006.
3. Thomson PA, et al. Association between the TRAX/DISC1 locus and both bipolar disorder and schizophrenia in the Scottish population. Molecular Psychiatry. 2005.
4. Bhatt DL, et al. GPR50 and energy homeostasis: Implications in obesity and metabolic disorders. Journal of Endocrinology. (General reference — specific article details require verification).

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