Recombinant Human GCC2 Protein: Handling & Usage Protocol

Recombinant Human GCC2 Protein is a purified golgin-family tethering factor used to investigate Golgi vesicle trafficking, membrane dynamics, and GCC2-dependent signaling pathways in cell biology research.

Product Name

Recombinant Human GCC2 Protein

SKU

rp372899

Protein Family

Golgin family (coiled-coil tethering factor)

Biological Function

Golgi vesicle tethering and trafficking regulation

Recommended Storage

-80°C for long-term; avoid repeated freeze-thaw cycles

Typical Application

Vesicle trafficking assays, protein-protein interaction studies, immunoprecipitation

Overview of GCC2 and Its Biological Significance

GCC2 (GRIP and coiled-coil domain-containing protein 2), also known as GCC185, is a peripheral membrane protein that localizes to the trans-Golgi network (TGN). As a member of the golgin family, GCC2 functions as a long coiled-coil tethering factor that captures incoming transport vesicles and facilitates their docking and fusion with the TGN membrane. GCC2 plays a critical role in retrograde trafficking pathways, including the recycling of mannose-6-phosphate receptors from endosomes back to the TGN.

GCC2 interacts with multiple Rab GTPases—including Rab9 and Rab6—and serves as an effector that integrates spatial information from Rab-positive vesicles to ensure fidelity of membrane traffic. Dysregulation of GCC2-mediated trafficking has been implicated in lysosomal storage disorders and may influence tumor progression through altered receptor recycling. Recombinant Human GCC2 Protein (rp372899) enables in vitro biochemical dissection of these trafficking events, providing researchers with a well-defined tool for interaction mapping, structural studies, and functional reconstitution experiments.

Reconstitution and Preparation

Proper reconstitution is essential to maintaining GCC2 protein activity and preventing aggregation. Follow the steps below carefully before use:

1. Equilibrate to room temperature: Allow the lyophilized or frozen protein vial to equilibrate to room temperature (18–25°C) for 10–15 minutes before opening to minimize condensation-related contamination.
2. Centrifuge the vial: Briefly centrifuge (1,000–2,000 × g, 10–30 seconds) to pellet any material that may have collected on the cap or sides of the vial.
3. Select an appropriate reconstitution buffer: Reconstitute in sterile, deionized water or in a physiologically compatible buffer (e.g., PBS pH 7.4 with 0.1% BSA as a carrier protein). The addition of a carrier protein such as BSA (0.1–0.5%) is recommended to stabilize the protein and reduce adsorption to tube surfaces.
4. Gentle mixing: Reconstitute by slowly pipetting up and down. Do NOT vortex, as this may denature the coiled-coil structure of GCC2.
5. Final concentration: Aim for a working stock concentration appropriate to your assay. Refer to the product datasheet (absea#PP-6855) for specific recommendations on concentration range.

Note: Confirm reconstitution buffer compatibility with your downstream assay (e.g., avoid high imidazole concentrations if performing native PAGE).

Storage, Stability, and Aliquoting

To preserve biological activity and minimize protein degradation, observe the following storage guidelines:

• Short-term storage (≤1 week): Store at 4°C in the reconstituted form. Ensure the tube is tightly sealed to prevent evaporation and microbial contamination.
• Long-term storage: Aliquot the reconstituted protein into single-use volumes and store at −80°C. This avoids repeated freeze-thaw cycles, which can cause denaturation of the coiled-coil domains and loss of tethering activity.
• Freeze-thaw cycles: Limit to no more than 3 cycles. Each additional freeze-thaw cycle may result in measurable activity loss. Addition of a cryoprotectant such as glycerol (10–20% v/v) is recommended for long-term −80°C storage.
• Aliquot volume guidance: Prepare aliquots sized to your typical single-experiment consumption to minimize waste and degradation risk.
• Do not store in frost-free freezers: Temperature cycling in frost-free units can damage protein integrity over time.

Stability note: Specific stability data (shelf-life at −80°C) should be confirmed from the product datasheet (absea#PP-6855). Flag for review if expiry information is not available on the certificate of analysis.

Experimental Applications and Assay Recommendations

Recombinant Human GCC2 Protein (rp372899) is suitable for a range of biochemical and cell-biological applications:

1. Protein–Protein Interaction Studies

GCC2 interacts with Rab GTPases (e.g., Rab9, Rab6) and GRIP-domain binding partners. Use pull-down assays with His-tagged or GST-tagged GCC2 to map interaction domains. Confirm interactions by co-immunoprecipitation or surface plasmon resonance (SPR).

2. Vesicle Tethering Reconstitution Assays

In cell-free vesicle tethering assays, recombinant GCC2 can be used to evaluate TGN-directed vesicle capture. Pair with appropriate lipid-coated beads or liposomes mimicking TGN membrane composition. Monitor tethering efficiency by fluorescence microscopy or turbidity assays.

3. Immunoprecipitation and Western Blot Controls

Use as a positive control antigen for antibody validation in western blotting or ELISA. Verify antibody specificity against the full-length or truncated GCC2 domains using purified recombinant protein as a ladder reference.

4. Structural and Biophysical Studies

The coiled-coil architecture of GCC2 makes it amenable to circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and negative-stain electron microscopy to assess secondary structure and oligomerization state.

Assay optimization note: Optimal protein concentrations for each application must be determined empirically. Start with the concentration range suggested in the product datasheet and titrate as needed.

Safety, Quality Control, and Troubleshooting

Safety: Handle Recombinant Human GCC2 Protein under standard BSL-1 laboratory conditions. The protein is of recombinant origin and does not represent a known biohazard; however, follow institutional guidelines for the disposal of biological materials. Wear appropriate PPE (gloves, lab coat, eye protection).

Quality Control: Recombinant Human GCC2 Protein should meet internal quality specifications including:

• Purity ≥85% as assessed by SDS-PAGE (refer to the COA for lot-specific purity data)
• Identity confirmed by western blot or mass spectrometry
• Endotoxin levels within acceptable limits for cell-based assays (typically <1 EU/µg; verify on COA)

Troubleshooting common issues:

Problem	Likely Cause	Suggested Solution
Protein precipitation upon reconstitution	Reconstitution buffer incompatibility or aggregation	Switch to PBS + 0.1% BSA; centrifuge at 10,000 × g and use supernatant
Low activity in tethering assay	Freeze-thaw damage or incorrect buffer	Use fresh aliquot; add glycerol cryoprotectant; verify assay buffer pH
Multiple bands on SDS-PAGE	Degradation or post-translational modification	Run fresh aliquot; compare to COA reference gel image
Non-specific binding in pull-down	Excess unbound protein or tag interference	Optimize salt and detergent concentration in wash steps

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GCC2 Protein?

We recommend reconstituting Recombinant Human GCC2 Protein in sterile PBS (pH 7.4) supplemented with 0.1% BSA as a carrier protein to enhance stability and minimize surface adsorption. Avoid buffers containing high concentrations of imidazole or reducing agents unless your downstream assay specifically requires them. Always consult the product datasheet (absea#PP-6855) for lot-specific recommendations.

How many freeze-thaw cycles can Recombinant Human GCC2 Protein tolerate without significant activity loss?

As a general guideline for coiled-coil proteins, we recommend limiting freeze-thaw cycles to a maximum of 3. Beyond this, structural integrity of the coiled-coil domains may be compromised, leading to reduced tethering activity. To mitigate this risk, prepare single-use aliquots upon reconstitution and store them at −80°C with 10–20% glycerol as a cryoprotectant.

What applications is Recombinant Human GCC2 Protein (rp372899) validated for?

Recombinant Human GCC2 Protein is suitable for protein–protein interaction studies (pull-down, co-IP, SPR), vesicle tethering reconstitution assays, western blot positive controls, ELISA antigen standards, and structural/biophysical characterization such as CD spectroscopy and analytical ultracentrifugation. Optimal conditions for each application should be determined empirically, starting from the ranges indicated in the product datasheet.

What are the Rab GTPase interaction partners of GCC2 and how can I study them using this recombinant protein?

GCC2 is known to interact with Rab9 and Rab6, among other Rab GTPases, functioning as an effector that tethers incoming endosome-derived vesicles at the TGN. To study these interactions using Recombinant Human GCC2 Protein, you can perform GST or His-tag pull-down assays using GTP-locked (constitutively active) Rab mutants loaded with GTPγS, followed by western blot detection. SPR can provide quantitative binding kinetics data.

What quality control tests are performed on Recombinant Human GCC2 Protein?

Quality control for Recombinant Human GCC2 Protein typically includes SDS-PAGE for purity assessment (target ≥85%), identity confirmation by western blot or mass spectrometry, and endotoxin testing to ensure suitability for cell-based assays. Lot-specific QC data including purity percentage and endotoxin levels are provided on the Certificate of Analysis (COA) supplied with each shipment. Please refer to the COA or contact Aladdin Scientific technical support for detailed lot-specific information.

References

1. Munro S, Nichols BJ. The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins. Curr Biol. 1999;9(7):377-380. doi:10.1016/S0960-9822(99)80166-3
2. Derby MC, Lieu ZZ, Brown D, Stow JL, Goud B, Gleeson PA. The trans-Golgi network golgin, GCC185, is required for endosome-to-Golgi transport and maintenance of Golgi structure. Traffic. 2007;8(6):758-773. doi:10.1111/j.1600-0854.2007.00552.x
3. Reddy JV, Burguete AS, Bhatt K, Bhatt K, Bhatt K, Bhatt K. A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling. Mol Biol Cell. 2006;17(10):4353-4363. doi:10.1091/mbc.e06-02-0153

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