Recombinant Human GPAT2 Protein: Production & Usage Protocol

Recombinant Human GPAT2 Protein is a research-grade enzyme used to study glycerophospholipid biosynthesis and lipid metabolism pathways in human biology.

Product Name

Recombinant Human GPAT2 Protein

SKU

rp367899

Protein Function

Catalyzes the initial and rate-limiting step of glycerophospholipid biosynthesis: acylation of sn-glycerol-3-phosphate

Gene Name

GPAT2 (Glycerol-3-Phosphate Acyltransferase 2, Mitochondrial)

Application Areas

Lipid metabolism research, enzyme activity assays, biochemical pathway studies

Storage Recommendation

Avoid repeated freeze-thaw cycles; store aliquots at -80°C for long-term stability

Reference Source

absea#PP-3131

Overview of GPAT2 and Its Biological Role

Glycerol-3-Phosphate Acyltransferase 2 (GPAT2) is a mitochondrial enzyme encoded by the GPAT2 gene in humans. It catalyzes the acylation of sn-glycerol-3-phosphate (G3P) with a long-chain acyl-CoA, producing lysophosphatidic acid (LPA). This reaction represents the first and rate-limiting committed step in the de novo biosynthesis of glycerophospholipids and triacylglycerols.

GPAT2 is expressed predominantly in the testis and has been associated with piRNA (PIWI-interacting RNA) biogenesis and spermatogenesis, in addition to its canonical role in lipid biosynthesis. Research into GPAT2 contributes to understanding lipid dysregulation in metabolic diseases and certain cancers. Recombinant Human GPAT2 Protein (rp367899) provides researchers with a reliable tool for in vitro enzymatic studies, inhibitor screening, and pathway investigation.

Materials and Equipment Required

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

• Recombinant Human GPAT2 Protein (rp367899, Aladdin Scientific)
• Appropriate reconstitution buffer (typically PBS or manufacturer-recommended buffer; verify with product datasheet)
• Sterile, nuclease-free water or buffer for reconstitution
• Microcentrifuge tubes (low-protein-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Benchtop microcentrifuge
• Dry ice or liquid nitrogen for transport and snap-freezing of aliquots
• -80°C freezer for long-term storage
• Spectrophotometer or BCA/Bradford assay reagents for protein concentration verification
• SDS-PAGE and Western blot equipment for purity and identity confirmation (optional but recommended)

All procedures should be conducted in a clean workspace to prevent contamination. Use personal protective equipment (PPE) including gloves and lab coat throughout the protocol.

Step-by-Step Reconstitution and Handling Protocol

Follow these steps carefully to reconstitute and handle Recombinant Human GPAT2 Protein for optimal activity and stability:

1. Retrieval from storage: Remove the vial from -80°C storage and allow it to thaw slowly on ice (approximately 10–15 minutes). Do not thaw at room temperature or use a water bath, as rapid temperature changes may compromise protein integrity.
2. Brief centrifugation: Pulse-spin the vial in a microcentrifuge (3,000–5,000 × g, 10–15 seconds) to collect all lyophilized material or liquid to the bottom of the tube.
3. Reconstitution: Add the recommended volume of sterile reconstitution buffer (refer to the product datasheet for specific buffer composition and volume). Gently pipette up and down to mix; do not vortex, as this may denature the protein.
4. Equilibration: Allow the reconstituted protein to stand on ice for 5 minutes to ensure complete dissolution.
5. Concentration verification: Measure protein concentration using BCA or Bradford assay and confirm against the stated lot concentration.
6. Aliquoting: Divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles, which significantly reduce enzymatic activity.
7. Storage of aliquots: Snap-freeze aliquots in liquid nitrogen and store at -80°C. For short-term use (up to 1 week), aliquots may be stored at 4°C.
8. Quality verification (recommended): Run a reducing SDS-PAGE to confirm molecular weight and purity before use in critical experiments.

Note: Specific buffer formulations, recommended concentrations, and exact molecular weight should be confirmed against the official product datasheet (absea#PP-3131). All figures are subject to lot-specific variation.

Experimental Applications and Assay Considerations

Recombinant Human GPAT2 Protein (rp367899) is suitable for a range of in vitro research applications, including but not limited to:

• Enzyme activity assays: GPAT2 activity can be measured by monitoring the transfer of a radiolabeled or fluorescently tagged acyl group from acyl-CoA to sn-glycerol-3-phosphate. Appropriate positive and negative controls must be included in every assay.
• Inhibitor and activator screening: The recombinant protein serves as a target for small-molecule screening campaigns aimed at identifying modulators of lipid biosynthesis relevant to metabolic disease or oncology research.
• Protein–protein interaction studies: Co-immunoprecipitation or pull-down assays using GPAT2 can help elucidate interaction partners within the lipid biosynthesis complex or piRNA pathway.
• Structural studies: Purified recombinant GPAT2 may be used as a substrate for crystallography or cryo-EM studies pending sufficient purity and yield.
• Antibody validation: Recombinant GPAT2 can serve as a positive control antigen in Western blot or ELISA-based antibody validation experiments.

Important considerations: Optimize reaction buffer pH, temperature, and cofactor concentrations empirically for each assay format. Enzymatic activity may be sensitive to detergent concentration, reducing agents, and metal ions. Always include appropriate controls and refer to peer-reviewed literature for established assay conditions.

Troubleshooting Common Issues

If unexpected results are obtained, consult the following troubleshooting guide:

• Low or no enzymatic activity: Verify that the protein has not been subjected to multiple freeze-thaw cycles. Check reconstitution buffer compatibility, substrate concentrations, and assay temperature. Confirm that cofactors (e.g., Mg²⁺, CoA derivatives) are present at appropriate concentrations.
• Protein aggregation or precipitation: Ensure the reconstitution buffer is at the correct pH and ionic strength. Centrifuge the reconstituted solution at low speed (10,000 × g, 5 minutes, 4°C) to remove aggregates before use.
• Unexpected bands on SDS-PAGE: Minor bands may represent glycosylation heterogeneity or minor degradation products. Confirm the major band at the expected molecular weight. If bands are absent, verify antibody specificity and protein loading.
• Poor reproducibility between experiments: Use freshly thawed single-use aliquots for each experiment. Standardize assay conditions including incubation time, temperature, and substrate lot. Record lot numbers for all reagents.
• Contamination signals: Use low-protein-binding tubes and tips. Include a no-protein negative control in every assay to identify background signals.

If problems persist, contact Aladdin Scientific technical support and provide the product SKU (rp367899), lot number, and a detailed description of the experimental conditions.

Frequently asked questions

What is the recommended storage condition for Recombinant Human GPAT2 Protein (rp367899)?

For long-term storage, aliquots of Recombinant Human GPAT2 Protein should be stored at -80°C. Repeated freeze-thaw cycles should be strictly avoided, as they significantly reduce protein activity and stability. For short-term use within one week, aliquots may be kept at 4°C.

What biological function does GPAT2 serve, and why is it relevant to research?

GPAT2 (Glycerol-3-Phosphate Acyltransferase 2) is a mitochondrial enzyme that catalyzes the first committed, rate-limiting step of glycerophospholipid and triacylglycerol biosynthesis by acylating sn-glycerol-3-phosphate. It is also implicated in piRNA biogenesis and spermatogenesis. Its roles in lipid metabolism make it relevant to metabolic disease, obesity, and cancer research.

How should I verify the quality of the recombinant protein before use in experiments?

Quality verification is recommended via reducing SDS-PAGE to confirm the expected molecular weight and assess purity. Protein concentration should be measured using a BCA or Bradford assay and compared against the lot-specific value stated in the datasheet. For functional assays, an enzyme activity test with appropriate controls is the most direct validation method.

Can Recombinant Human GPAT2 Protein be used for inhibitor screening assays?

Yes, Recombinant Human GPAT2 Protein (rp367899) is suitable for in vitro inhibitor and activator screening assays targeting the glycerophospholipid biosynthesis pathway. Researchers should empirically optimize substrate and protein concentrations, buffer conditions, and detection methods for their specific screening platform. Appropriate positive and negative controls must always be included.

What should I do if I observe protein aggregation after reconstitution?

If aggregation occurs, first verify that the reconstitution buffer pH and ionic strength are correct per the product datasheet. Centrifuge the solution at 10,000 × g for 5 minutes at 4°C to pellet aggregates, then carefully transfer the supernatant for use. Avoid vortexing and ensure the protein is thawed gently on ice to minimize aggregation.

References

1. absea Product Reference PP-3131: Recombinant Human GPAT2 Protein. Absea Biotechnology Ltd.
2. Wendel AA, Lewin TM, Coleman RA. Glycerol-3-phosphate acyltransferases: rate limiting enzymes of triacylglycerol biosynthesis. Biochim Biophys Acta. 2009;1791(6):501-506. doi:10.1016/j.bbalip.2008.10.010
3. Shiromoto Y, Kuramochi-Miyagawa S, Daiba A, et al. GPAT2, mitochondria-localized protein, is required for piRNA biogenesis. RNA. 2013;19(12):1680-1689. doi:10.1261/rna.038521.113

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