Recombinant Human GPATCH3 Protein: Expression & Use Protocol

Recombinant Human GPATCH3 Protein is a research-grade reagent designed to support investigations into G-patch domain biology, RNA processing pathways, and protein–protein interaction studies in human cellular systems.

Product Name

Recombinant Human GPATCH3 Protein

SKU

rp374329

Protein Family

G-patch domain-containing protein family

Species Origin

Human (Homo sapiens)

Primary Application

RNA processing research, protein interaction assays, biochemical studies

Storage Recommendation

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

Background and Biological Function of GPATCH3

GPATCH3 (G Patch Domain-Containing Protein 3) is a member of the G-patch superfamily, characterized by a conserved glycine-rich motif of approximately 50 amino acids known as the G-patch domain. G-patch proteins are predominantly associated with RNA metabolism, acting as cofactors that activate RNA helicases of the DEAH/RHA family, thereby regulating pre-mRNA splicing, ribosome biogenesis, and other RNA processing events.

In humans, GPATCH3 is expressed across multiple tissues and has been implicated in the modulation of RNA helicase activity. Its conserved G-patch domain is believed to mediate direct physical interactions with partner helicases, stimulating their ATPase and unwinding activities. Research into GPATCH3 and related G-patch proteins contributes to understanding RNA surveillance mechanisms and may have relevance to disease contexts where RNA processing is dysregulated.

Materials and Equipment Required

• Recombinant Human GPATCH3 Protein (SKU: rp374329)
• Sterile, nuclease-free water or recommended reconstitution buffer (e.g., PBS, pH 7.4, with carrier protein if required)
• Microcentrifuge tubes (low-protein-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Benchtop microcentrifuge
• Dry ice or liquid nitrogen for snap-freezing aliquots
• -20°C and -80°C freezers
• SDS-PAGE and Western blot apparatus (for purity verification)
• Spectrophotometer or BCA/Bradford assay kit (for protein quantification)
• Relevant assay buffers appropriate to downstream application (e.g., interaction assay, ATPase assay)

Step-by-Step Reconstitution and Handling Protocol

Step 1 – Thawing: 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 to minimize protein degradation.

Step 2 – Brief Centrifugation: Gently centrifuge the vial at low speed (≤1000 × g, 30 seconds) to collect any material from the cap and walls before opening.

Step 3 – Reconstitution: If supplied in lyophilized form, reconstitute by adding the appropriate volume of sterile, nuclease-free water or the supplier-recommended buffer to achieve the desired working concentration. Add the reconstitution solution slowly along the vial wall and gently swirl. Avoid vigorous vortexing, which may denature the protein.

Step 4 – Equilibration: Allow the reconstituted protein to stand on ice for 5 minutes to ensure complete dissolution.

Step 5 – Aliquoting: Prepare single-use aliquots in low-binding microcentrifuge tubes to prevent repeated freeze-thaw cycles, which degrade protein activity and purity.

Step 6 – Storage: Store aliquots at -80°C for long-term use or at -20°C for short-term use (up to 1 month). Once thawed, keep on ice and use within the same working session.

Step 7 – Quality Verification: Prior to use in critical assays, verify protein integrity by SDS-PAGE and confirm concentration by BCA or Bradford assay.

Recommended Applications and Assay Guidelines

Recombinant Human GPATCH3 Protein is suitable for a range of biochemical and cell-biology applications, including but not limited to:

• Protein–Protein Interaction Studies: Use in pull-down assays or co-immunoprecipitation experiments to identify and validate interaction partners, particularly DEAH-box RNA helicases.
• RNA Helicase Stimulation Assays: Evaluate the capacity of GPATCH3 to stimulate ATPase or unwinding activity of cognate helicase partners in vitro.
• Antibody Validation: Serve as a positive control antigen for Western blot or ELISA assays targeting GPATCH3.
• Structural Studies: Suitable as a component in biophysical characterization experiments such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC).

Optimize protein concentration and buffer conditions for each specific application. Inclusion of appropriate positive and negative controls is strongly recommended in all experimental setups.

Troubleshooting and Quality Considerations

Protein Precipitation: If precipitation is observed upon reconstitution, gently centrifuge at low speed and use only the supernatant. Precipitation may result from improper thawing, incorrect buffer conditions, or protein aggregation during storage. Ensure the reconstitution buffer is at the correct pH and ionic strength.

Low Activity: Reduced functional activity may indicate protein denaturation due to improper freeze-thaw handling. Always thaw on ice and avoid exposing the protein to room temperature for extended periods. Check that all buffers are freshly prepared and free from contaminating proteases.

Unexpected Bands on SDS-PAGE: Minor bands may represent post-translational modification variants, degradation products, or host-cell protein co-purification. If band pattern deviates significantly from the expected molecular weight, contact Aladdin Scientific technical support.

Contamination with Nucleases: As GPATCH3 is an RNA-associated protein, ensure that all buffers and plasticware are nuclease-free when performing RNA-related assays to avoid RNA substrate degradation.

Frequently asked questions

What is the recommended storage condition for Recombinant Human GPATCH3 Protein?

For long-term storage, keep the protein at -80°C. For short-term use within one month, -20°C is acceptable. Single-use aliquots should be prepared to avoid repeated freeze-thaw cycles, which can compromise protein integrity and activity.

In which types of assays can Recombinant Human GPATCH3 Protein be used?

Recombinant Human GPATCH3 Protein is suitable for protein–protein interaction studies (e.g., pull-down, co-IP), RNA helicase stimulation assays, antibody validation as a positive control antigen, and biophysical characterization experiments such as SPR or ITC. Optimal working concentrations should be determined empirically for each application.

How should I reconstitute Recombinant Human GPATCH3 Protein if it is supplied in lyophilized form?

Add the appropriate volume of sterile, nuclease-free water or supplier-recommended buffer (such as PBS, pH 7.4) slowly along the vial wall to reach the desired concentration. Gently swirl to mix; do not vortex vigorously. Allow the solution to stand on ice for 5 minutes before use to ensure complete dissolution.

What should I do if I observe precipitation after reconstitution?

If precipitation occurs, briefly centrifuge the vial at low speed (≤1000 × g) and collect only the supernatant for use. Check that the reconstitution buffer has the correct pH and ionic strength. If the problem persists, contact Aladdin Scientific technical support for assistance.

Why is it important to use nuclease-free materials when working with GPATCH3?

GPATCH3 is an RNA-associated protein involved in RNA processing pathways. In assays involving RNA substrates, any contaminating nucleases in buffers or plasticware could degrade the RNA, leading to unreliable results. Using certified nuclease-free water, buffers, and consumables ensures experimental integrity.

References

1. Bohnsack MT, Martin R, Granneman S, Ruprecht M, Schleiff E, Tollervey D. Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis. Mol Cell. 2009;36(4):583-592. doi:10.1016/j.molcel.2009.09.039
2. Robert-Paganin J, Rety S, Leulliot N. Regulation of DEAH/RHA helicases by G-patch proteins. Biomed Res Int. 2015;2015:931857. doi:10.1155/2015/931857
3. Studer MK, Ivanovic L, Weber ME, Marti S, Jonas S. Molecular basis for DEAH-helicase activation by G-patch proteins. Proc Natl Acad Sci USA. 2020;117(13):7159-7170. doi:10.1073/pnas.1913880117

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