Recombinant Human GBP2 Protein: Handling & Usage Protocol

Recombinant Human GBP2 Protein is a purified, biologically active GTPase that plays a central role in interferon-gamma-mediated innate immune responses and is suitable for a range of in vitro biochemical and cell-based assays.

Product Name

Recombinant Human GBP2 Protein

SKU

rp365216

Protein Family

Guanylate-Binding Protein (GBP) family, dynamin-like GTPase superfamily

Primary Biological Function

Mediates interferon-gamma (IFN-γ)-induced innate immune defense; exhibits GTPase activity hydrolyzing GTP to GDP and GMP

Recommended Storage

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

Typical Applications

GTPase activity assays, pull-down/co-immunoprecipitation, SDS-PAGE/Western blot, cell-based functional studies

Background & Biological Significance of GBP2

Guanylate-Binding Protein 2 (GBP2) is a member of the dynamin-like GTPase superfamily encoded by an interferon-stimulated gene (ISG). GBP2 is potently induced by interferon-gamma (IFN-γ) and, to a lesser extent, by type I interferons, placing it at the intersection of innate immune signaling and host defense against intracellular pathogens including bacteria, viruses, and parasites.

GBP2 functions by hydrolyzing GTP sequentially to GDP and GMP, a hallmark activity that distinguishes GBPs from classical GTPases. Upon activation, GBP2 undergoes isoprenylation at its C-terminal CaaX motif, facilitating membrane targeting and the formation of supramolecular assemblies on pathogen-containing vacuoles. These assemblies contribute to inflammasome activation, pyroptosis, and direct antimicrobial effector functions.

Research applications of GBP2 span immunology, infectious disease, oncology (GBP2 has been associated with tumor immune evasion and metastasis suppression), and structural biology. Recombinant Human GBP2 Protein (rp365216) enables researchers to study these mechanisms in controlled, defined biochemical conditions.

Materials & Equipment Required

Before beginning, ensure the following materials and equipment are available:

• Recombinant Human GBP2 Protein (Aladdin Scientific, SKU: rp365216)
• Sterile, nuclease-free water or recommended reconstitution buffer (e.g., PBS, pH 7.4, containing 0.1% BSA as carrier protein)
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Calibrated micropipettes and sterile filtered tips
• Benchtop microcentrifuge
• Dry ice or liquid nitrogen for aliquoting
• -80 °C and -20 °C freezers
• SDS-PAGE apparatus and Western blot equipment (for QC verification)
• GTPase activity assay kit (e.g., malachite green phosphate detection) if performing enzymatic assays
• Protein quantification reagents (Bradford or BCA assay)

Step-by-Step Reconstitution & Storage Protocol

Step 1 – Equilibration. Remove the vial of lyophilized Recombinant Human GBP2 Protein from storage and allow it to equilibrate to room temperature for 5–10 minutes before opening. This minimizes condensation inside the vial.

Step 2 – Reconstitution. Reconstitute the lyophilized protein in sterile PBS (pH 7.4) supplemented with 0.1% (w/v) BSA, or as specified in the product datasheet, to a stock concentration typically in the range of 0.1–1.0 mg/mL. Add the reconstitution buffer gently along the inner wall of the vial to prevent foaming. Do not vortex; mix by gentle pipetting or slow rotation.

Step 3 – Clarification. Centrifuge briefly (1,000 × g, 1 minute, 4 °C) to collect any residual material from the walls. Inspect visually for complete dissolution.

Step 4 – Quantification. Determine protein concentration using a BCA or Bradford assay with BSA standards, or by absorbance at 280 nm using the theoretical extinction coefficient for GBP2.

Step 5 – Aliquoting. Divide the reconstituted protein into single-use aliquots using low-binding tubes. Aliquot volumes should reflect individual experimental requirements to minimize freeze-thaw cycles.

Step 6 – Storage. Snap-freeze aliquots on dry ice or in liquid nitrogen and store at -80 °C for long-term use (up to 12 months recommended). For short-term use within 1–2 weeks, aliquots may be kept at -20 °C. Do NOT store at 4 °C for extended periods, as proteolytic degradation may occur.

Note: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided.

Experimental Applications & Assay Guidelines

GTPase Activity Assay: Recombinant Human GBP2 Protein is suitable for in vitro GTPase activity measurements. Prepare reaction mixtures containing GBP2 at appropriate concentrations in assay buffer (e.g., 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM DTT). Initiate the reaction by adding GTP substrate. Quantify inorganic phosphate release using a malachite green colorimetric kit at the desired time points. Include a no-enzyme control and a GTP-only blank.

SDS-PAGE & Western Blot (Quality Verification): Resolve 0.5–2 µg of reconstituted GBP2 on a 10–12% SDS-PAGE gel under reducing conditions. The expected molecular weight of GBP2 is approximately 67–73 kDa. Confirm identity by Western blot using a validated anti-GBP2 antibody.

Pull-Down & Co-Immunoprecipitation: GBP2 interacts with multiple immune effectors including IRGB10 and components of the NLRP3 inflammasome pathway. Use the recombinant protein as bait or prey in GST or His-tag pull-down systems with appropriate cell lysates or recombinant partner proteins.

Cell-Based Studies: When supplementing cell culture media, filter-sterilize the protein solution through a 0.2 µm membrane and confirm sterility. Ensure the carrier protein (BSA) concentration is compatible with the cell line and assay type being used.

Troubleshooting Common Issues

Low or Absent GTPase Activity: Verify that GTP substrate is fresh and not hydrolyzed during storage. Confirm MgCl₂ is present in the assay buffer; Mg²⁺ is essential for GTPase catalysis. Check that the protein was not subjected to repeated freeze-thaw cycles. Ensure the protein concentration used is within the linear detection range of the assay.

Unexpected Molecular Weight on SDS-PAGE: GBP2 may appear as a doublet or slightly higher-than-expected band due to post-translational modifications (e.g., isoprenylation) if expressed in a eukaryotic system, or as a non-modified form if expressed in E. coli. Confirm the production host from the datasheet and adjust expectations accordingly.

Protein Precipitation After Reconstitution: Ensure the buffer pH is within the recommended range. Addition of 5–10% glycerol to the storage buffer can stabilize the protein. If aggregation persists, centrifuge at higher speed (10,000 × g, 10 minutes, 4 °C) and collect the supernatant for use.

Weak Western Blot Signal: Increase protein loading or optimize antibody concentration and incubation conditions. Confirm antibody species compatibility with the recombinant human GBP2 sequence used.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GBP2 Protein?

The protein is generally reconstituted in sterile PBS (pH 7.4) supplemented with 0.1% (w/v) BSA as a carrier protein to enhance stability. Always consult the specific product datasheet (rp365216) for the exact recommended conditions, as formulation may vary by lot.

How should I store Recombinant Human GBP2 Protein to maintain its activity?

Lyophilized protein should be stored at -20 °C to -80 °C. After reconstitution, divide into single-use aliquots and store at -80 °C for long-term stability. Avoid repeated freeze-thaw cycles, which progressively denature the protein and reduce GTPase activity. Short-term storage at -20 °C (1–2 weeks) is acceptable for aliquots in active use.

What applications is Recombinant Human GBP2 Protein validated for?

Recombinant Human GBP2 Protein (rp365216) is suitable for in vitro GTPase activity assays, SDS-PAGE purity analysis, Western blotting, protein-protein interaction studies (pull-down, co-IP), and cell-based functional studies related to innate immunity and interferon signaling. Please verify specific validated applications on the product datasheet prior to use.

Why does GBP2 have an unusually large molecular weight compared with typical GTPases?

GBP2 belongs to the dynamin-like GTPase superfamily, which is characterized by a large N-terminal GTPase domain combined with a helical middle domain and a GED (GTPase effector domain). This multi-domain architecture results in a molecular weight of approximately 67–73 kDa, significantly larger than the small Ras-family GTPases (~20–25 kDa).

Can Recombinant Human GBP2 Protein be used in cell-free expression or structural studies?

Yes, purified recombinant GBP2 can be used as a reference standard or interaction partner in cell-free systems and has been used in structural biology studies to investigate the conformational changes associated with GTP binding and hydrolysis. Ensure protein purity and homogeneity are verified by SDS-PAGE before use in biophysical or structural assays.

References

1. Praefcke GJK. Regulation of innate immune functions by guanylate-binding proteins. Int J Med Microbiol. 2018;308(1):237-245. doi:10.1016/j.ijmm.2017.10.013
2. Kim BH, et al. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense. Nat Immunol. 2016;17(5):481-489.
3. Vestal DJ, Jeyaratnam JA. The guanylate-binding proteins: emerging insights into the biochemical properties and functions of this family of large interferon-induced guanosine triphosphatases. J Interferon Cytokine Res. 2011;31(1):89-97. doi:10.1089/jir.2010.0105

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