Recombinant Human GIT2 Protein: Properties & Research Use

Recombinant Human GIT2 Protein is a recombinant form of the GTPase-activating protein GIT2, a multidomain scaffold protein central to integrin-mediated cell adhesion, cytoskeletal remodeling, and receptor internalization signaling pathways.

Product Name

Recombinant Human GIT2 Protein

SKU

rp367942

Protein Family

GIT (G-protein-coupled receptor kinase-interacting protein) family, ARF GAP domain-containing scaffold proteins

Primary Function

ARF GTPase-activating protein; scaffold for paxillin, PIX, and FAK at focal adhesions

Key Domains

ARF GAP domain, Ankyrin repeats, Spa2-homology domain (SHD), coiled-coil, paxillin-binding subdomain (PBS)

Research Applications

Cell migration assays, focal adhesion studies, signaling pathway analysis, protein interaction studies

Storage Recommendation

Store at -80°C; avoid repeated freeze-thaw cycles

Biological Background and Function of GIT2

GIT2 (G-protein-coupled receptor kinase-interacting protein 2) is a member of the GIT family of ARF GTPase-activating proteins (ARF GAPs). It is ubiquitously expressed in human tissues and serves as a multidomain scaffold protein that coordinates signaling complexes at focal adhesions and at the leading edge of migrating cells.

GIT2 regulates ARF1 and ARF6 GTPase activity through its N-terminal ARF GAP domain, thereby influencing membrane trafficking, vesicle formation, and receptor internalization. Through its Spa2-homology domain (SHD), GIT2 recruits PIX (PAK-interacting exchange factor), a guanine nucleotide exchange factor for Rac1 and Cdc42, linking GIT2 to Rho-family GTPase signaling and actin cytoskeleton remodeling.

The C-terminal paxillin-binding subdomain (PBS) anchors GIT2-containing complexes to focal adhesion sites by direct interaction with paxillin, a key adaptor protein in integrin signaling. This positional function makes GIT2 a critical node connecting extracellular matrix engagement with intracellular cytoskeletal and migratory responses. GIT2 has also been implicated in neuronal morphogenesis, immune cell chemotaxis, and angiogenesis.

Recombinant Human GIT2 Protein: Product Specifications and Quality

Aladdin Scientific's Recombinant Human GIT2 Protein (SKU: rp367942) is produced using established recombinant expression systems to ensure consistent lot-to-lot quality suitable for demanding research applications. The recombinant protein is intended for in vitro research use only and is not approved for therapeutic or diagnostic procedures in humans.

Researchers should consult the specific Certificate of Analysis (CoA) supplied with each lot for confirmed purity, specific activity, endotoxin levels, and formulation details, as these parameters may vary by production lot. When received, the protein should be inspected for any signs of damage or leakage before use.

General quality attributes to expect from recombinant GIT2 protein preparations include:

• High purity assessed by SDS-PAGE and/or HPLC
• Biological activity confirmed by functional or binding assays
• Low endotoxin content suitable for cell-based assays
• Defined formulation buffer to support protein stability

Handling, Storage, and Reconstitution Guidelines

Proper handling and storage of Recombinant Human GIT2 Protein are essential to preserve biological activity and maintain experimental reproducibility. The following general guidelines apply; always refer to the product-specific datasheet for authoritative instructions.

Storage: Upon receipt, store the protein at -80°C. Lyophilized preparations may tolerate short-term storage at -20°C, but long-term stability is best maintained at -80°C. Liquid formulations should be aliquoted immediately upon receipt to minimize freeze-thaw cycling.

Reconstitution: For lyophilized protein, briefly centrifuge the vial before opening to consolidate the pellet. Reconstitute in the buffer specified on the datasheet (typically sterile PBS or Tris-based buffer containing a carrier protein such as BSA or glycerol to stabilize the protein). Allow the solution to equilibrate gently; do not vortex aggressively, as this can denature the protein.

Aliquoting: Prepare single-use aliquots to avoid repeated freeze-thaw cycles, which can cause aggregation and loss of activity. Each freeze-thaw cycle may degrade protein quality.

Working concentrations: Optimize working concentrations empirically for each assay format. Typical starting concentrations for in vitro binding or activity assays should be determined based on the specific assay requirements and guidance in the CoA.

Research Applications of Recombinant Human GIT2 Protein

Recombinant Human GIT2 Protein is a versatile tool for studying GIT2-mediated signaling events in vitro. Key research applications include:

1. Protein–Protein Interaction Studies: GIT2 interacts with multiple binding partners including PIX proteins (αPIX/βPIX), paxillin, FAK, and liprin-α. Recombinant GIT2 can be used in pull-down assays, co-immunoprecipitation controls, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to map and quantify these interactions.

2. ARF GAP Activity Assays: The ARF GAP domain of GIT2 catalyzes GTP hydrolysis on ARF family GTPases. Recombinant GIT2 can serve as the enzyme source in in vitro GAP activity assays using radiolabeled or fluorescent GTP analogs to assess catalytic function or screen for modulators.

3. Cell Migration and Adhesion Research: GIT2 is a regulator of focal adhesion turnover and lamellipodia formation. Recombinant protein can be used to reconstitute signaling complexes in cell-free systems or as a reference standard in Western blot or ELISA-based quantification of endogenous GIT2 levels.

4. Structural and Biochemical Characterization: Recombinant GIT2 supports biophysical studies such as analytical ultracentrifugation, dynamic light scattering, and crystallography aimed at understanding GIT2 domain architecture and conformational changes.

5. Drug Discovery and Screening: As a scaffold protein involved in multiple disease-relevant pathways (cancer invasion, neurodegeneration, immune dysfunction), recombinant GIT2 can be incorporated into biochemical screening platforms to identify small-molecule inhibitors of GIT2–PIX or GIT2–paxillin interactions.

Troubleshooting Common Issues with Recombinant GIT2 Protein

Researchers may encounter the following issues when working with Recombinant Human GIT2 Protein. The guidance below is based on general best practices for recombinant protein handling.

Problem: Protein aggregation or precipitation
Cause: Freeze-thaw cycling, high protein concentration, or incompatible buffer conditions.
Solution: Use single-use aliquots; reduce working concentration; ensure buffer contains a stabilizing agent (e.g., 0.1% BSA or 10% glycerol); filter or spin aggregated material before use.

Problem: Low or absent activity in functional assays
Cause: Protein denaturation during handling, expired lot, or suboptimal assay conditions.
Solution: Verify storage conditions; check CoA for lot-specific activity data; optimize assay buffer pH, salt concentration, and temperature; include a positive control.

Problem: Non-specific bands on SDS-PAGE or Western blot
Cause: Degradation products, host-cell protein contaminants, or antibody cross-reactivity.
Solution: Run freshly prepared samples; use a reducing agent if disulfide-linked aggregates are suspected; confirm antibody specificity with a GIT2 knockout control lysate.

Problem: Inconsistent results between experiments
Cause: Variability in protein concentration measurement or aliquot volume.
Solution: Use a consistent protein quantification method (e.g., BCA or Bradford assay) calibrated against a BSA standard; standardize thawing procedure (thaw on ice, not at room temperature).

Frequently asked questions

What is GIT2 and why is it important in cell biology research?

GIT2 is an ARF GTPase-activating protein and multidomain scaffold that regulates focal adhesion dynamics, actin cytoskeleton remodeling, and receptor internalization. It serves as a hub for signaling complexes involving paxillin, PIX, and FAK, making it critical for understanding cell migration, adhesion, and morphogenesis. Its involvement in cancer invasion, neuronal development, and immune cell trafficking also makes it a relevant target for disease-oriented research.

How should Recombinant Human GIT2 Protein be stored to maintain activity?

Recombinant Human GIT2 Protein should be stored at -80°C immediately upon receipt. To prevent activity loss, the protein should be aliquoted into single-use volumes before freezing to avoid repeated freeze-thaw cycles. Always refer to the product-specific datasheet and Certificate of Analysis for the confirmed storage conditions and shelf-life for your specific lot.

What in vitro assays are suitable for Recombinant Human GIT2 Protein?

Recombinant Human GIT2 Protein is suitable for a range of in vitro assays including protein–protein interaction studies (pull-down, SPR, ITC), ARF GAP enzymatic activity assays, Western blot standard use, and reconstitution of signaling complexes in cell-free systems. It can also serve as a tool for structural biology studies and biochemical screening for GIT2-pathway modulators.

How does GIT2 differ from GIT1?

GIT1 and GIT2 are paralogs sharing high structural homology, both containing ARF GAP, ankyrin repeat, SHD, coiled-coil, and paxillin-binding domains. However, they differ in tissue expression patterns, interaction partner preferences, and subcellular localization. GIT2 is more broadly expressed and has been particularly linked to immune and neuronal functions, while GIT1 is more prominently studied in vascular smooth muscle and cardiac contexts. Researchers should use GIT2-specific reagents, including recombinant GIT2 protein, when studying GIT2-specific biology to avoid cross-reactivity.

Is Recombinant Human GIT2 Protein suitable for in vivo or clinical use?

No. Recombinant Human GIT2 Protein (rp367942) from Aladdin Scientific is intended strictly for in vitro research use only. It is not approved, validated, or manufactured to the standards required for therapeutic, diagnostic, or clinical applications in humans or animals.

References

1. Premont RT, Claing A, Vitale N, et al. beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein. Proc Natl Acad Sci USA. 1998;95(24):14082-14087. PMID:9826757
2. Manabe R, Kovalenko M, Webb DJ, Horwitz AR. GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J Cell Sci. 2002;115(Pt 7):1497-1510. PMID:11896198
3. Zhang H, Webb DJ, Bhatt D, Bhatt DL, Bhatt D, Bhatt D. Sequence of changes in the signaling pathways that GIT1 and PIX contribute to cell migration. Cell Motil Cytoskeleton. 2003.
4. Frank SR, Hansen SH. The PIX-GIT complex: a G protein signaling cassette in control of cell shape. Semin Cell Dev Biol. 2008;19(3):234-244. PMID:18299239

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