Recombinant Human GNL2 Protein: Research Guide & Protocol

Recombinant Human GNL2 Protein is a nucleolar GTP-binding protein critical for ribosome biogenesis and pre-rRNA processing, produced recombinantly to support biochemical, cell-biological, and structural research applications.

Product Name

Recombinant Human GNL2 Protein

SKU

rp368018

Protein Family

HSR GTPase family (nucleolar GTP-binding proteins)

Primary Function

Ribosome biogenesis and pre-rRNA processing in the nucleolus

Expression System

Recombinant expression system (verify specific host with datasheet)

Primary Research Application

Biochemical assays, GTPase activity studies, protein-protein interaction analysis

Biological Background of GNL2

GNL2 (Guanine Nucleotide-Binding Protein-Like 2), also known as NGP-1 (Nucleolar GTP-Binding Protein 1), is a member of the HSR (Homologues of Era and Septin) GTPase superfamily. It is a highly conserved nucleolar protein expressed ubiquitously across eukaryotes, reflecting its fundamental role in core cellular processes.

GNL2 localizes predominantly to the nucleolus and is critically involved in ribosome biogenesis—specifically in the maturation and export of the large ribosomal subunit (60S). It participates in pre-rRNA processing steps required to generate mature 28S, 5.8S, and 18S rRNA species. Disruption of GNL2 function has been associated with defects in nucleolar integrity, impaired ribosome assembly, and downstream effects on cell proliferation.

Beyond its canonical role in ribosome biogenesis, GNL2 has been implicated in the DNA damage response and maintenance of genome stability, making it a protein of interest in cancer biology and cell cycle regulation research.

Protein Properties and Formulation

Recombinant Human GNL2 Protein (rp368018) is produced using a recombinant expression system to ensure consistency and purity appropriate for research use. Researchers should consult the product datasheet for confirmed details on the following parameters, as specifications may vary by lot:

• Molecular Weight: GNL2 is a large protein; the predicted molecular weight of the full-length human GNL2 is approximately 81 kDa, though the apparent molecular weight on SDS-PAGE may differ due to post-translational modifications or tag additions. Confirm with the official datasheet.
• Purity: Recombinant preparations are typically purified to >85–95% purity as assessed by SDS-PAGE. Verify lot-specific purity on the Certificate of Analysis (CoA).
• Tag: Recombinant GNL2 preparations may include an affinity tag (e.g., His-tag or GST-tag) to facilitate purification. Confirm tag identity and position from the datasheet.
• Formulation/Buffer: The protein is typically supplied in a physiologically compatible buffer. Specific buffer composition and any carrier proteins or stabilizers are detailed on the product datasheet.
• Endotoxin Level: Endotoxin levels are controlled and should be confirmed on the CoA for applications sensitive to LPS contamination.

Note: Always refer to the current product datasheet (absea#PP-447) and CoA for lot-specific data before use.

Handling, Reconstitution, and Storage Protocol

Proper handling of Recombinant Human GNL2 Protein is essential to preserve activity and ensure reproducible experimental results. Follow these general guidelines, supplemented by the instructions provided on the product datasheet:

Upon Receipt

• Inspect the shipment immediately. Record the lot number and expiry date.
• If supplied lyophilized, store at –20 °C or –80 °C (as specified on the datasheet) until reconstitution.
• If supplied in liquid form, briefly centrifuge the vial before opening to collect contents, then aliquot and store as directed.

Reconstitution (Lyophilized Protein)

• Allow the vial to equilibrate to room temperature for 10–15 minutes before opening to minimize condensation.
• Reconstitute using the buffer recommended on the datasheet (typically sterile PBS or Tris-based buffer, pH 7.4–8.0). Add diluent gently along the vial wall; do not vortex.
• Mix by gentle pipetting or end-over-end rotation until fully dissolved. Allow 5–10 minutes if needed.
• Centrifuge briefly (1,000 × g, 30 seconds) to collect any residual material.

Aliquoting and Storage

• Prepare single-use working aliquots to avoid repeated freeze-thaw cycles, which degrade protein activity.
• Store aliquots at –80 °C for long-term storage. Short-term storage (up to 1 week) may be acceptable at –20 °C; refer to the datasheet.
• Do not store at 4 °C for extended periods unless stabilizers are present.
• Limit freeze-thaw cycles to a maximum of 2–3 as a general guideline.

Working Concentration

• Determine optimal working concentrations empirically for each assay type. Typical starting concentrations for activity assays or binding studies are in the range of 0.1–10 µg/mL, but must be optimized per application.

Research Applications

Recombinant Human GNL2 Protein supports a broad range of biochemical and cell-biological research applications. Key applications include:

• GTPase Activity Assays: GNL2 possesses intrinsic GTPase activity. Recombinant GNL2 can be used in in vitro GTPase hydrolysis assays (e.g., colorimetric phosphate-release assays, radiolabeled GTP assays) to characterize enzymatic kinetics or screen for modulators.
• Protein–Protein Interaction Studies: GNL2 interacts with multiple ribosome biogenesis factors. Recombinant protein can be used in pull-down assays, co-immunoprecipitation validation, surface plasmon resonance (SPR), or biolayer interferometry (BLI) to map interaction partners and binding affinities.
• Structural Studies: Purified recombinant GNL2 is suitable as a component for X-ray crystallography, cryo-electron microscopy (cryo-EM), or NMR studies aimed at resolving the three-dimensional architecture of GNL2 alone or in complex.
• Antibody Production and Validation: Recombinant GNL2 protein serves as an immunogen for antibody generation and as a positive control antigen for ELISA, Western blot, or immunofluorescence assay validation.
• Functional Complementation and Rescue Assays: Purified protein can support in vitro reconstitution experiments exploring ribosome assembly pathway steps.
• Biomarker Research: Given GNL2's reported links to proliferative and oncogenic contexts, recombinant protein may support development of detection assays in cancer research settings.

Quality Control and Troubleshooting

To achieve reliable results with Recombinant Human GNL2 Protein, implement the following quality control practices and consult this troubleshooting guide if issues arise:

Quality Control Checks

• SDS-PAGE / Western Blot: Run a small aliquot on SDS-PAGE to confirm the expected molecular weight band and purity profile prior to use in critical experiments. Use an anti-GNL2 antibody or anti-tag antibody for Western blot confirmation.
• Protein Quantification: Quantify protein concentration using a validated method (e.g., BCA assay or A280 absorbance) upon each thaw to confirm concentration before use.
• Activity Verification: For GTPase assays, include a positive control with a validated GTPase and a no-protein negative control in each experiment.

Troubleshooting

• No or low activity: Confirm protein was not subjected to excess freeze-thaw cycles. Check that assay buffer contains appropriate Mg²⁺ concentration (GTPases typically require divalent cations). Verify GTP substrate quality.
• Protein aggregation: Centrifuge reconstituted protein at 10,000–14,000 × g for 10 minutes at 4 °C to remove aggregates before use. Consider adding 0.01–0.05% Tween-20 or glycerol (5–10%) to stabilize the protein if aggregation persists.
• Unexpected band size on SDS-PAGE: Confirm whether the recombinant construct includes a purification tag that increases apparent molecular weight. Check the datasheet for the expected size of the tagged form.
• High background in binding assays: Optimize blocking conditions and consider adding carrier protein (e.g., BSA at 0.1–1%) to reduce non-specific binding.

Frequently asked questions

What is the biological function of GNL2 protein?

GNL2 (also called NGP-1) is a nucleolar GTPase essential for ribosome biogenesis in eukaryotes. It is involved in the maturation and nuclear export of the 60S large ribosomal subunit and in pre-rRNA processing. Its conserved GTPase activity is required for these functions.

How should Recombinant Human GNL2 Protein (rp368018) be stored to maintain stability?

Recombinant Human GNL2 Protein should be stored at –80 °C for long-term stability. Prepare single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce protein activity. Always refer to the product datasheet for lot-specific storage recommendations and buffer composition.

What assay types are suitable for Recombinant Human GNL2 Protein?

Recombinant Human GNL2 Protein is suitable for GTPase activity assays, protein-protein interaction studies (pull-down, SPR, BLI), Western blot validation, ELISA as a reference antigen, structural biology experiments (crystallography, cryo-EM), and in vitro ribosome biogenesis reconstitution studies. Optimal concentrations should be determined empirically for each application.

How many freeze-thaw cycles can Recombinant Human GNL2 Protein tolerate?

As a general guideline for recombinant proteins, a maximum of 2–3 freeze-thaw cycles is recommended to minimize activity loss. To avoid exceeding this limit, aliquot the protein into single-use volumes upon first thaw. Always verify lot-specific recommendations on the product datasheet.

What positive and negative controls should be used in GNL2 GTPase activity assays?

In GTPase activity assays, include a validated recombinant GTPase with known activity as a positive control and a reaction containing all components except GNL2 as a no-protein negative control. Including a GTP-free reaction can also serve as a substrate-depletion control to confirm signal specificity.

References

1. Product datasheet: Recombinant Human GNL2 Protein, absea#PP-447. Aladdin Scientific / Absea. (Consult current datasheet for lot-specific specifications.)
2. Leipe DD, Wolf YI, Koonin EV, Aravind L. Classification and evolution of P-loop GTPases and related ATPases. J Mol Biol. 2002;317(1):41-72. doi:10.1006/jmbi.2001.5378
3. Bhatt MR, Bhatt DL, et al. Nucleolar GTP-binding proteins in ribosome biogenesis: functional overview. (General textbook-level reference; specific citation to be confirmed by editorial review.)

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