Recombinant Human GPKOW Protein: Research Guide & Protocol

Recombinant Human GPKOW Protein is a purified recombinant form of the human G-patch domain and KOW motif-containing protein, widely used in RNA splicing, spliceosome regulation, and related molecular biology research applications.

Product Name

Recombinant Human GPKOW Protein

SKU

rp365503

Protein Function

Involved in pre-mRNA splicing and spliceosome regulation via its G-patch and KOW domains

Expression System

Recombinant expression system (see product datasheet for specific host details)

Recommended Storage

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

Primary Research Applications

RNA splicing assays, protein-protein interaction studies, spliceosome biochemistry

Background: GPKOW Protein Biology

GPKOW (G-patch domain and KOW motifs-containing protein), also known as T54 or MOS2 homolog, is a nuclear protein encoded by the GPKOW gene in humans. It contains a G-patch domain — a conserved glycine-rich motif found in a variety of RNA-associated proteins — and one or more KOW (Kyprides, Ouzounis, Woese) motifs that mediate nucleic acid and protein interactions.

GPKOW plays an established role in pre-mRNA splicing. It interacts with components of the spliceosome, particularly with the RNA helicase DHX16 (also known as hPRP2), and is thought to act as a co-activator or regulatory factor that stimulates helicase activity during spliceosome remodeling steps. Disruption of GPKOW function has been associated with defects in splicing fidelity, underscoring its biological importance.

Understanding GPKOW function has implications for basic RNA biology, as well as for disease contexts where aberrant splicing contributes to pathology, including cancer and genetic disorders.

Protein Specifications and Quality

Recombinant Human GPKOW Protein (SKU: rp365503) is produced using recombinant expression technology to ensure consistent batch-to-batch quality. Researchers should consult the official product datasheet for lot-specific information including:

• Purity: Assessed by SDS-PAGE and/or HPLC; typical recombinant research-grade proteins are ≥85% pure (verify on current datasheet).
• Formulation: Supplied in a carrier buffer; formulation details including buffer composition and any stabilizing additives are listed on the datasheet.
• Molecular Weight: Corresponds to the predicted molecular weight of the GPKOW isoform expressed, potentially altered by any fusion tag; confirm from datasheet.
• Endotoxin Level: Tested and reported on the certificate of analysis (CoA).
• Activity: Functional validation data, if available, are provided in the product documentation.

Always request the CoA for the specific lot received to confirm conformance to specifications before use in critical experiments.

Reconstitution and Handling Protocol

Proper handling of Recombinant Human GPKOW Protein is essential to preserve activity and ensure reproducible results. Follow these general guidelines, supplemented by any lot-specific instructions on the datasheet:

1. Thawing: Thaw the protein on ice or at 4°C. Do not thaw at room temperature or use a water bath at elevated temperatures, as this may cause aggregation or denaturation.
2. Reconstitution (lyophilized product): If supplied in lyophilized form, allow the vial to equilibrate to room temperature briefly before opening. Reconstitute in sterile, ultrapure water or the buffer specified on the datasheet. Add solvent gently along the vial wall and allow to dissolve without vortexing. Centrifuge briefly at low speed to collect contents.
3. Concentration: After reconstitution, determine working concentration by absorbance (A280) or a protein assay (e.g., BCA or Bradford) referenced against a BSA standard.
4. Aliquoting: Divide reconstituted protein into single-use aliquots to minimize freeze-thaw cycles. Snap-freeze aliquots in liquid nitrogen prior to storage at -80°C.
5. Carrier Protein Addition: For dilute working solutions (<10 µg/mL), consider adding BSA (0.1%) to prevent adsorption to tube surfaces.

Always work on ice during handling and minimize the time the protein spends at room temperature.

Storage Conditions and Stability

Long-term storage integrity of Recombinant Human GPKOW Protein depends on strict adherence to recommended conditions:

• Long-term storage: Store at -80°C in single-use aliquots for maximum stability.
• Short-term storage: If the protein will be used within 1–2 weeks, it may be stored at -20°C; however, -80°C is preferred.
• Avoid repeated freeze-thaw cycles: Each freeze-thaw cycle can reduce protein activity and promote aggregation. Pre-aliquoting before freezing is strongly recommended.
• Working solution: Prepared working dilutions should be used promptly and not stored for extended periods. If short-term storage of diluted protein is necessary, hold at 4°C for no more than 24–48 hours.
• Stability monitoring: Assess protein integrity periodically by SDS-PAGE if long-term storage exceeds recommended timeframes.

Refer to the product datasheet for expiration date and any specific stability data generated under accelerated or real-time conditions.

Recommended Research Applications

Recombinant Human GPKOW Protein is suited to a range of in vitro and biochemical research applications, including but not limited to:

• RNA Splicing Assays: Use as a recombinant component in reconstituted splicing reactions to study spliceosome assembly, activation, or catalysis.
• Protein–Protein Interaction Studies: Employ in pull-down assays, co-immunoprecipitation (co-IP), or surface plasmon resonance (SPR) experiments to map GPKOW interactome partners, particularly spliceosomal helicases such as DHX16.
• Protein–RNA Interaction Studies: Use in electrophoretic mobility shift assays (EMSA) or RNA immunoprecipitation (RIP) setups to characterize GPKOW binding to specific RNA substrates.
• Helicase Stimulation Assays: Investigate the G-patch domain-mediated stimulation of partner helicase unwinding activity in defined biochemical systems.
• Antibody Validation: Serve as a positive control antigen for validating GPKOW-targeting antibodies in Western blot or ELISA formats.
• Structural Studies: Support biophysical characterization including circular dichroism (CD) or limited proteolysis experiments to probe domain structure.

Optimal working concentrations and assay conditions should be determined empirically for each specific application.

Frequently asked questions

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

The reconstitution buffer is specified on the product datasheet and certificate of analysis for each lot. In general, sterile ultrapure water or a compatible physiological buffer (e.g., PBS) is appropriate for initial reconstitution of lyophilized recombinant proteins; however, always follow the lot-specific instructions provided with the product to avoid activity loss.

How many freeze-thaw cycles can Recombinant Human GPKOW Protein withstand?

As with most recombinant proteins, repeated freeze-thaw cycles are detrimental to GPKOW protein integrity and activity. It is strongly recommended to aliquot the protein into single-use volumes immediately after reconstitution and to store aliquots at -80°C. This minimizes freeze-thaw events and helps maintain protein quality throughout the project.

What quality controls are performed on Recombinant Human GPKOW Protein (rp365503)?

Aladdin Scientific performs standard quality control testing on recombinant proteins, which typically includes purity assessment by SDS-PAGE and/or HPLC, endotoxin testing, and identity confirmation. Lot-specific results are documented in the certificate of analysis (CoA) available upon request. Researchers should review the CoA before use in sensitive experiments.

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

Recombinant Human GPKOW Protein is primarily validated for in vitro biochemical applications such as binding assays, helicase stimulation studies, and antibody validation. Its suitability for cell-based assays depends on the experimental context and delivery method. Researchers intending to use it in cellular systems should confirm endotoxin levels from the CoA and optimize delivery conditions empirically.

Where can I find the GPKOW protein product datasheet and certificate of analysis?

The product datasheet for Recombinant Human GPKOW Protein (rp365503) is available on the Aladdin Scientific website product page. The certificate of analysis (CoA) for your specific lot can be requested from Aladdin Scientific customer service or downloaded directly from the website using your lot number.

References

1. Yoshimoto R, Kataoka N, Okawa K, Ohno M. Identification and characterization of SF3b155 as a target of the splicing factor GPKOW. Nucleic Acids Research. 2009.
2. Hamann F, Enders M, Ficner R. Structural basis for RNA translocation by DEAH-box ATPases. Nucleic Acids Research. 2019.
3. Studer MK, Ivanovic L, Weber ME, Marti S, Jonas S. Structural basis for DEAH-helicase activation by G-patch proteins. Proceedings of the National Academy of Sciences. 2020.

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