Recombinant Human GON7 Protein: Preparation & Usage Protocol

Recombinant Human GON7 Protein is a purified recombinant subunit of the KEOPS complex designed to support biochemical interaction studies, complex reconstitution experiments, and functional assays requiring a well-characterized human GON7 component.

Product Name

Recombinant Human GON7 Protein

SKU

rp367990

Protein Function

Subunit of the KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) complex, involved in tRNA modification and telomere maintenance

Species Origin

Human (Homo sapiens)

Application

Biochemical assays, protein-protein interaction studies, KEOPS complex reconstitution

Storage Recommendation

Store lyophilized protein at -20°C or -80°C; avoid repeated freeze-thaw cycles

Background: GON7 and the KEOPS Complex

GON7 (also known as C14orf142) is a small accessory subunit of the evolutionarily conserved KEOPS complex, which also comprises LAGE3, OSGEP, TP53RK, and TPRKB. The KEOPS complex catalyzes the threonylcarbamoylation of adenosine at position 37 (t6A modification) of ANN-decoding tRNAs, a universally conserved modification essential for translational fidelity and efficiency.

Beyond its role in tRNA modification, the KEOPS complex has been implicated in telomere maintenance, transcriptional regulation, and DNA repair. GON7 is believed to stabilize the complex and facilitate its assembly, making it an important target for studies of genetic diseases associated with KEOPS dysfunction, including Galloway-Mowat syndrome.

Reconstitution Protocol

Materials Required:

• Recombinant Human GON7 Protein (rp367990)
• Sterile, deionized water or phosphate-buffered saline (PBS, pH 7.4)
• Carrier protein solution (e.g., 0.1% BSA in PBS) if required for stability
• Microcentrifuge tubes (low-binding recommended)
• Ice

Step-by-Step Reconstitution:

1. Allow the lyophilized vial to equilibrate to room temperature for 10–15 minutes before opening to prevent moisture condensation on the protein.
2. Briefly centrifuge the vial at low speed (300–500 × g, 30 seconds) to pellet any residual lyophilized material from the walls and cap.
3. Reconstitute the protein by adding the recommended volume of sterile PBS (pH 7.4) or deionized water directly to the vial. Gently swirl or pipette to mix; do not vortex.
4. Allow the reconstituted protein to stand on ice for 5 minutes to ensure complete dissolution.
5. Prepare single-use aliquots to minimize freeze-thaw cycles and store at -80°C for long-term use or at -20°C for short-term use (up to 1 month).

Note: Consult the product-specific datasheet for the exact recommended reconstitution volume and concentration. Flag for review if no datasheet value is available.

Storage and Stability Guidelines

Proper storage of Recombinant Human GON7 Protein is critical to maintaining its biological activity and structural integrity:

• Lyophilized form: Store at -20°C or -80°C in a dry environment. Under these conditions, the protein is stable for at least 12 months from the date of manufacture.
• Reconstituted form: Store working aliquots at -80°C. Avoid repeated freeze-thaw cycles, as each cycle may result in protein aggregation and loss of activity. Use a freshly thawed aliquot for each experiment.
• Working solutions: Once thawed, keep on ice during the experiment and use within the same working day. Do not re-freeze reconstituted protein that has been used in an assay.
• Carrier protein: Addition of a carrier protein such as 0.1% BSA may improve stability in dilute solutions, particularly at concentrations below 10 µg/mL.

Note: Specific stability data for this lot should be confirmed against the Certificate of Analysis (CoA). Flag for review if lot-specific data are unavailable.

Experimental Applications and Tips

Recombinant Human GON7 Protein is suitable for several research applications. The following guidance helps ensure optimal experimental outcomes:

1. Protein–Protein Interaction Studies: GON7 can be used in pull-down assays or co-immunoprecipitation experiments to investigate its interactions with other KEOPS complex subunits (LAGE3, OSGEP, TP53RK, TPRKB). Optimize salt and detergent conditions to preserve native interactions.

2. KEOPS Complex Reconstitution: When reconstituting the full KEOPS complex in vitro, combine stoichiometric amounts of each subunit and allow sufficient incubation time at 4°C for complex assembly. Verify assembly by size-exclusion chromatography or native PAGE.

3. Biochemical and Enzymatic Assays: Use appropriate positive and negative controls in each assay run. Titrate protein concentrations to determine the linear range of detection for your specific assay format.

4. General Tips:

• Always use low-binding tubes and pipette tips to minimize protein loss due to surface adsorption.
• Centrifuge reconstituted protein at 10,000 × g for 5 minutes at 4°C prior to use to remove any aggregates.
• Record lot number and reconstitution date on each aliquot for traceability.

Quality Control and Troubleshooting

Quality Control: Recombinant Human GON7 Protein from Aladdin Scientific (rp367990) is manufactured and tested to ensure consistent purity and performance. Each lot is typically analyzed by SDS-PAGE and/or Western blot to confirm molecular weight and purity. Refer to the Certificate of Analysis (CoA) provided with each shipment for lot-specific QC data.

Common Issues and Troubleshooting:

Issue	Possible Cause	Recommended Action
Protein precipitation or turbidity after reconstitution	Aggregation due to improper reconstitution or temperature shock	Allow vial to reach room temperature before reconstitution; reconstitute gently; centrifuge to remove aggregates
Loss of activity	Repeated freeze-thaw cycles or prolonged storage at -20°C	Use single-use aliquots; store at -80°C for long-term
No signal in binding assay	Insufficient protein concentration or incompatible buffer conditions	Titrate protein; optimize buffer pH and salt concentration
Non-specific bands on Western blot	Cross-reactivity or high antibody concentration	Optimize antibody dilution; include appropriate controls

Frequently asked questions

What is the recommended reconstitution volume for Recombinant Human GON7 Protein (rp367990)?

The recommended reconstitution volume is specified on the product datasheet and Certificate of Analysis (CoA) supplied with each lot. As a general guideline, reconstitute lyophilized protein in sterile PBS (pH 7.4) or deionized water to yield a stock concentration of at least 100 µg/mL. Always refer to the lot-specific CoA for accurate instructions.

How should I store Recombinant Human GON7 Protein to maintain activity?

Store the lyophilized protein at -20°C or -80°C in a dry environment until reconstitution. After reconstitution, prepare single-use aliquots and store at -80°C for long-term use. Avoid repeated freeze-thaw cycles, as these can lead to protein aggregation and significant loss of biological activity.

What applications is Recombinant Human GON7 Protein suitable for?

Recombinant Human GON7 Protein is suitable for protein-protein interaction studies, KEOPS complex reconstitution in vitro, pull-down assays, co-immunoprecipitation, and biochemical characterization experiments. It may also be used as an antigen for antibody validation. Confirm suitability for your specific application with the product datasheet.

What is the role of GON7 in the KEOPS complex?

GON7 is a small accessory subunit of the KEOPS complex that is thought to contribute to complex stability and assembly. The KEOPS complex as a whole is responsible for the t6A (threonylcarbamoyladenosine) modification of tRNAs at position 37, which is essential for accurate and efficient protein translation. Mutations in KEOPS subunits, including GON7, have been linked to Galloway-Mowat syndrome.

How can I verify the purity and integrity of my GON7 protein lot?

Each lot of Recombinant Human GON7 Protein is accompanied by a Certificate of Analysis (CoA) detailing purity (typically assessed by SDS-PAGE), identity, and quality control test results. Run an SDS-PAGE or Western blot with the reconstituted protein to confirm the expected molecular weight and purity before use in sensitive downstream assays.

References

1. Wan LC, Mao DY, Neculai D, et al. Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Research. 2013.
2. Braun DA, Shril S, Sinha A, et al. Mutations in WDR4 as a new cause of Galloway-Mowat syndrome. American Journal of Human Genetics. 2018.
3. Arrondel C, Missoury S, Snoek R, et al. Defects in t6A tRNA modification due to GON7 and YRDC mutations lead to Galloway-Mowat syndrome. Nature Communications. 2019.

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