Recombinant Human GPATCH8 Protein: Handling & Usage Protocol

Recombinant Human GPATCH8 Protein is a G-patch domain-containing recombinant protein intended for use in biochemical characterization, protein–protein interaction studies, and RNA-processing research applications.

Product Name

Recombinant Human GPATCH8 Protein

SKU

rp375065

Protein Family

G-patch domain-containing proteins (GPATCH family)

Recommended Storage

-20°C or -80°C; avoid repeated freeze-thaw cycles

Primary Applications

Biochemical assays, protein interaction studies, RNA-processing research

Host/Expression System

To be confirmed with product datasheet (see review flag)

Background: GPATCH8 and the G-Patch Domain Family

GPATCH8 (G-patch domain-containing protein 8) is a member of the G-patch protein superfamily, characterized by a conserved glycine-rich motif known as the G-patch domain. G-patch domain proteins are found predominantly in eukaryotes and are recognized as regulators of RNA metabolism, functioning as cofactors that activate DEAH-box RNA helicases involved in pre-mRNA splicing, ribosome biogenesis, and other RNA-processing pathways.

GPATCH8 has been identified in the nucleus, where it is proposed to interact with components of the spliceosome or associated RNA remodeling complexes. The precise biological role of GPATCH8 remains an active area of investigation, making recombinant GPATCH8 protein a valuable reagent for studying its molecular interactions and biochemical activities.

Reconstitution and Storage Protocol

Materials required: Sterile deionized water or the buffer specified on the product datasheet, low-binding microcentrifuge tubes, and a benchtop microcentrifuge.

1. Equilibrate the vial: Allow the lyophilized or frozen protein vial to equilibrate to room temperature for 5–10 minutes before opening to minimize condensation-related contamination. Centrifuge briefly (1,000 × g, 30 seconds) to collect material at the bottom of the vial.
2. Reconstitute: Add the volume of sterile reconstitution buffer recommended on the product datasheet. Gently pipette up and down; do not vortex. Allow to dissolve at room temperature for 10 minutes, then clarify by centrifugation at 10,000 × g for 5 minutes at 4°C.
3. Aliquot: Divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles, which can lead to protein aggregation and loss of activity.
4. Storage: Store working aliquots at -20°C for up to 1 month or at -80°C for longer-term storage. Refer to the product datasheet for the specific recommended conditions for rp375065.

Note: Specific buffer composition, concentration, and storage duration must be verified against the official product datasheet for rp375065. See review flags.

Experimental Applications and Assay Guidance

Recombinant Human GPATCH8 Protein is suitable for the following research applications. Optimal conditions should be determined empirically for each assay system.

• Protein–Protein Interaction Studies (co-IP, GST pull-down, SPR): Use GPATCH8 protein to probe interactions with DEAH-box helicases (e.g., DHX15, DHX16) or other spliceosomal components. Ensure appropriate negative controls are included.
• Helicase Stimulation Assays: G-patch proteins are known to stimulate DEAH-box RNA helicase activity. GPATCH8 can be used in ATPase or RNA unwinding assays to characterize its co-factor function.
• Western Blot Positive Control: Reconstituted GPATCH8 protein can serve as a molecular weight and antibody specificity control in western blot experiments.
• ELISA Standard: May be used as a calibration standard when developing GPATCH8-specific immunoassays, subject to purity verification.

Always perform a pilot titration experiment to determine the optimal protein concentration for each application. Protein activity and binding characteristics may vary depending on assay buffer composition and temperature.

Quality Control and Troubleshooting

Quality control checks: Before using the protein in critical experiments, confirm identity and purity by SDS-PAGE and, where possible, by western blot using a validated anti-GPATCH8 antibody. The expected band should correspond to the predicted molecular weight of GPATCH8; verify the exact expected MW from the product datasheet for rp375065.

Common issues and solutions:

• Precipitation after reconstitution: Centrifuge at 10,000–16,000 × g for 10 minutes at 4°C and use only the supernatant. Ensure reconstitution buffer is pre-warmed to room temperature.
• Loss of activity upon storage: Avoid multiple freeze-thaw cycles. Add carrier protein (e.g., 0.1% BSA) if storing dilute solutions, as recommended by the datasheet.
• Non-specific bands on western blot: Verify antibody cross-reactivity; use the recombinant protein as a positive molecular weight marker alongside experimental lysates.
• Poor binding in pull-down assays: Optimize salt concentration, pH, and detergent content of the binding buffer. Reduce protein concentration to minimize non-specific interactions.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GPATCH8 Protein (rp375065)?

The specific reconstitution buffer, volume, and final concentration are detailed on the official product datasheet for rp375065. As a general guideline for recombinant proteins, sterile PBS (pH 7.4) supplemented with a carrier protein such as 0.1% BSA is commonly used; however, always defer to the product-specific datasheet for accurate instructions.

How should Recombinant Human GPATCH8 Protein be stored to maintain maximum activity?

Reconstituted protein should be aliquoted into single-use volumes and stored at -80°C for long-term preservation, or at -20°C for up to one month. Repeated freeze-thaw cycles should be strictly avoided as they promote protein aggregation and activity loss. Adding a carrier protein such as BSA is recommended when working with dilute concentrations.

What experimental applications is Recombinant Human GPATCH8 Protein validated for?

Recombinant Human GPATCH8 Protein is intended for applications including protein–protein interaction studies (co-immunoprecipitation, pull-down assays), helicase stimulation assays, western blot positive controls, and ELISA standard development. Specific validated applications are listed on the product datasheet for rp375065; users should confirm suitability for their particular assay system.

How does GPATCH8 function as part of the G-patch protein family?

GPATCH8 contains a conserved G-patch domain, which is a hallmark of proteins that act as cofactors for DEAH-box RNA helicases. G-patch proteins stimulate helicase ATPase and RNA unwinding activities, thereby regulating RNA remodeling events such as pre-mRNA splicing and ribosome biogenesis. The precise role of GPATCH8 within this family is still under active investigation.

Can Recombinant Human GPATCH8 Protein be used in cell-based assays?

Recombinant Human GPATCH8 Protein is primarily intended for in vitro biochemical applications. Use in cell-based assays may require additional optimization, including consideration of protein delivery methods and potential immunogenicity. Consult the product datasheet and relevant literature before designing cell-based experiments.

References

1. Bohnsack KE, Bohnsack MT. Uncovering the assembly pathway of human ribosomes and its emerging links to disease. EMBO J. 2019;38(13):e100781. doi:10.15252/embj.2018100781
2. Studer MK, Ivanovic L, Weber ME, Marti S, Jonas S. Structural basis for DEAH-helicase activation by G-patch proteins. Proc Natl Acad Sci USA. 2020;117(12):7159-7170. doi:10.1073/pnas.1913880117
3. Aladdin Scientific. Recombinant Human GPATCH8 Protein, SKU rp375065. Product Datasheet.

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