Recombinant Human GAN Protein: Research Guide & Protocol

Recombinant Human GAN Protein is a research-grade recombinant form of gigaxonin, the E3 ubiquitin ligase adaptor encoded by the GAN gene, essential for regulating intermediate filament turnover and implicated in giant axonal neuropathy.

Product Name

Recombinant Human GAN Protein

SKU

rp375079

Gene Name

GAN (Gigaxonin)

Protein Function

E3 ubiquitin ligase adaptor; regulates intermediate filament protein degradation

Disease Relevance

Giant Axonal Neuropathy (GAN); autosomal recessive neurodegenerative disorder

Recommended Storage

Store at -80°C; avoid repeated freeze-thaw cycles (general guideline; verify with CoA)

Biological Background of GAN Protein

The GAN gene encodes gigaxonin, a BTB-Kelch domain-containing protein that functions as a substrate adaptor for the Cullin-3 (CUL3) E3 ubiquitin ligase complex. Gigaxonin targets multiple intermediate filament (IF) proteins — including neurofilament light (NEFL), vimentin, and GFAP — for ubiquitin-mediated proteasomal degradation, thereby maintaining cytoskeletal homeostasis in neurons and other cell types.

Loss-of-function mutations in GAN cause Giant Axonal Neuropathy (GAN), a rare autosomal recessive neurodegenerative disease characterised by the progressive accumulation of intermediate filaments within axons, leading to axonal swelling, motor and sensory deficits, and premature death. The GAN protein is therefore a critical node connecting the ubiquitin-proteasome system (UPS) with cytoskeletal quality control.

Protein Properties and Formulation

Recombinant Human GAN Protein (SKU: rp375079) is produced using recombinant expression systems to yield a functionally relevant form of gigaxonin suitable for in vitro research applications. Key considerations for working with this protein include:

• Purity: Research-grade; specific purity and endotoxin values are provided on the Certificate of Analysis (CoA) supplied with each lot. Researchers should verify lot-specific data before use.
• Formulation: Supplied in a stabilising buffer; exact composition is stated on the product CoA. Avoid exposure to detergents that may disrupt Kelch domain folding.
• Molecular Weight: Gigaxonin has a predicted molecular weight of approximately 66 kDa (full-length human); recombinant constructs may vary depending on tags or truncations — confirm with CoA.
• Activity: Suitable for binding assays, pull-down experiments, and structural studies; functional ubiquitin ligase adaptor activity should be validated in the user's specific assay format.

Handling, Reconstitution, and Storage Protocol

Proper handling of Recombinant Human GAN Protein is essential to preserve activity and integrity. Follow the general guidelines below, and always refer to the lot-specific CoA for authoritative instructions:

1. Receipt: Upon arrival, immediately transfer the protein to the recommended storage temperature. Inspect packaging for integrity and record the lot number.
2. Reconstitution (if lyophilised): Briefly centrifuge the vial before opening to collect lyophilised material. Reconstitute with the volume of sterile, ultra-pure water or the buffer specified in the CoA. Allow the solution to stand for 5–10 minutes at room temperature and mix gently by pipetting — do not vortex.
3. Aliquoting: Prepare single-use aliquots immediately after reconstitution to prevent activity loss from repeated freeze-thaw cycles. Recommended aliquot volume depends on anticipated experimental use.
4. Storage: Store aliquots at −80 °C for long-term stability. For short-term use (≤1 week), storage at 4 °C in the presence of a carrier protein (e.g., 0.1% BSA) may be acceptable — verify with CoA.
5. Working Concentrations: Titrate the protein in your specific assay to determine the optimal working concentration. Typical in vitro binding assays employ recombinant proteins in the nanomolar-to-micromolar range.

Research Applications

Recombinant Human GAN Protein supports a wide range of research applications in neuroscience, cell biology, and drug discovery:

• Protein–Protein Interaction Studies: Investigate gigaxonin's interactions with CUL3, KLHDC proteins, and intermediate filament substrates (e.g., NEFL, vimentin, GFAP) using pull-down, co-immunoprecipitation, or surface plasmon resonance (SPR) assays.
• Ubiquitination Assays: Use as an adaptor component in reconstituted CUL3-RBX1-GAN E3 ligase assays to study substrate ubiquitination kinetics in vitro.
• Structural Biology: Suitable as an antigen for antibody generation or as a standard in Western blot analyses; may also support cryo-EM or X-ray crystallography studies of BTB-Kelch domain complexes.
• Disease Modelling: Supports mechanistic studies of Giant Axonal Neuropathy pathogenesis, useful for validating cellular models or assessing potential therapeutic interventions targeting the GAN pathway.
• Biomarker and Drug Discovery Research: Useful as a screening tool to identify small molecules that modulate gigaxonin-substrate interactions or stabilise gigaxonin function.

Frequently asked questions

What is the GAN protein and why is it important in research?

GAN (gigaxonin) is a BTB-Kelch domain E3 ubiquitin ligase adaptor that regulates intermediate filament protein degradation via the Cullin-3 complex. Loss-of-function mutations cause Giant Axonal Neuropathy, making GAN a key target for understanding cytoskeletal homeostasis, neurodegeneration mechanisms, and potential gene therapy approaches.

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

As a general guideline, store the protein at −80 °C in single-use aliquots to avoid repeated freeze-thaw cycles. Always refer to the Certificate of Analysis (CoA) supplied with your specific lot for authoritative storage and stability information, as formulation may vary between lots.

What assay formats are suitable for Recombinant Human GAN Protein?

Recombinant Human GAN Protein is suitable for pull-down assays, co-immunoprecipitation, surface plasmon resonance (SPR), in vitro ubiquitination assays, Western blot standards, and as an antigen for antibody validation. Researchers should optimise protein concentrations empirically within their specific experimental system.

What is the molecular weight of the recombinant GAN protein?

Full-length human gigaxonin has a predicted molecular weight of approximately 66 kDa. However, the exact observed molecular weight of the recombinant product may differ due to expression tags, truncations, or post-translational modifications. Always confirm with the CoA provided for your specific lot.

Can Recombinant Human GAN Protein be used for drug screening applications?

Yes. Recombinant Human GAN Protein can serve as a tool for identifying small molecules that modulate gigaxonin-substrate interactions or restore gigaxonin function. It is particularly relevant for researchers developing therapeutic strategies for Giant Axonal Neuropathy. Assay conditions should be optimised and validated by the end user.

References

1. Bomont P, Cavalier L, Blondeau F, et al. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nature Genetics. 2000;26(3):370-374. doi:10.1038/81701
2. Mahammad S, Murthy SN, Didonna A, et al. Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation. Journal of Clinical Investigation. 2013;123(5):1964-1975. doi:10.1172/JCI66387
3. Scrivo A, Codogno P, Bomont P. Gigaxonin E3 ligase governs ATG16L1 turnover to control autophagosome production. Nature Communications. 2019;10(1):780. doi:10.1038/s41467-019-08331-w

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