Recombinant Human ATL1 Protein: Handling & Application Protocol
Recombinant Human ATL1 Protein: Handling & Application Protocol
Recombinant Human ATL1 Protein is a research-grade GTPase essential for endoplasmic reticulum membrane fusion studies and hereditary spastic paraplegia (HSP) mechanistic investigations.
- Product Name
- Recombinant Human ATL1 Protein
- SKU
- rp369093
- Protein Function
- GTPase involved in endoplasmic reticulum membrane fusion and tubular ER network formation
- Disease Relevance
- Mutations linked to Hereditary Spastic Paraplegia (HSP) type SPG3A
- Recommended Storage
- -80°C for long-term storage; avoid repeated freeze-thaw cycles
- Typical Applications
- GTPase activity assays, ER fusion assays, protein interaction studies, Western blot, and ELISA
Background: ATL1 Biology and Research Significance
Atlastin-1 (ATL1), encoded by the ATL1 gene, is a dynamin-related GTPase that localizes primarily to the endoplasmic reticulum (ER) membrane. ATL1 plays a critical role in mediating homotypic membrane fusion events required for the formation and maintenance of the tubular ER network, particularly in neuronal axons.
Loss-of-function mutations in ATL1 are among the most common causes of early-onset Hereditary Spastic Paraplegia (HSP) subtype SPG3A, a progressive neurodegenerative disorder characterized by lower-limb spasticity. Recombinant Human ATL1 Protein enables biochemical dissection of GTPase catalytic activity, membrane tethering mechanisms, and pathogenic mutant behavior in vitro, supporting drug discovery and mechanistic neuroscience research.
Materials Required
- Recombinant Human ATL1 Protein (SKU: rp369093)
- Sterile deionized water or manufacturer-recommended reconstitution buffer
- Microcentrifuge tubes (low-binding, 1.5 mL)
- Pipettes and calibrated tips
- Refrigerated microcentrifuge
- GTP or non-hydrolyzable GTP analogs (e.g., GTPγS) for activity assays
- SDS-PAGE and Western blot reagents (for QC verification)
- Appropriate assay buffers (e.g., 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl₂)
- BSA protein standards for concentration determination
Note: Always handle recombinant proteins on ice to minimize activity loss. Consult the product datasheet for lot-specific concentration and buffer composition details.
Reconstitution and Storage Protocol
- Thaw the lyophilized or frozen Recombinant Human ATL1 Protein on ice for 10–15 minutes. Do not thaw at room temperature.
- Centrifuge briefly (1,000 × g, 30 seconds) to collect any material from the tube walls and cap before opening.
- Reconstitute the protein by adding the recommended volume of sterile deionized water or reconstitution buffer dropwise to achieve the desired working concentration. Gently pipette to mix; do not vortex.
- Aliquot the reconstituted protein into single-use volumes appropriate for your assay to avoid repeated freeze-thaw cycles, which can reduce protein integrity and GTPase activity.
- Storage: Store aliquots at −80°C for long-term use (up to 12 months is typical for recombinant proteins; verify with lot-specific datasheet). Short-term working stocks may be kept at 4°C for up to 48 hours.
- Quality check: Run an SDS-PAGE gel on the first use of each new lot to confirm molecular weight and purity before proceeding with functional assays.
Important: Add a carrier protein (e.g., 0.1% BSA) to diluted working solutions if protein adsorption to tube surfaces is a concern at low concentrations.
Experimental Applications and Assay Guidelines
GTPase Activity Assay
ATL1's intrinsic GTPase activity can be measured using a malachite green phosphate release assay or a coupled enzyme assay (pyruvate kinase/lactate dehydrogenase). Incubate ATL1 protein with GTP in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl₂) at 37°C and monitor inorganic phosphate release over time. Include appropriate negative controls (no protein, no GTP) and a positive control GTPase if available.
Liposome Fusion Assay
Recombinant ATL1 can be reconstituted into proteoliposomes to evaluate its membrane tethering and fusion activity in vitro using fluorescence-based lipid mixing or content-mixing assays. Follow established lipid reconstitution protocols suited to the GTPase concentration and lipid composition used.
Protein–Protein Interaction Studies
ATL1 interacts with ER-shaping proteins such as reticulons and DP1/Yop1p homologs. Use pull-down assays or surface plasmon resonance (SPR) to characterize these interactions. Recombinant Human ATL1 Protein is suitable as bait or prey in co-immunoprecipitation setups.
Western Blot and ELISA
Recombinant ATL1 can serve as a positive control or standard in immunodetection assays. Verify antibody specificity and linear detection range prior to quantitative experiments.
Troubleshooting and Best Practices
| Issue | Possible Cause | Recommended Action |
|---|---|---|
| Low or absent GTPase activity | Protein denaturation due to improper storage or freeze-thaw | Use freshly thawed aliquots; confirm protein integrity by SDS-PAGE |
| Unexpected band size on SDS-PAGE | Degradation or aggregation | Check lot-specific datasheet for expected MW; spin sample at 10,000 × g before loading |
| High background in activity assay | Phosphate contamination in buffers or reagents | Use phosphate-free buffers; prepare fresh GTP solution |
| Poor liposome reconstitution efficiency | Detergent concentration too high or lipid ratio suboptimal | Optimize detergent removal steps; verify lipid composition |
| Non-reproducible results between lots | Lot-to-lot variation in specific activity | Normalize by active fraction; run activity assay on every new lot |
Always consult the Aladdin Scientific product datasheet for lot-specific purity, concentration, and activity data before initiating experiments.
Frequently asked questions
What is the recommended reconstitution buffer for Recombinant Human ATL1 Protein?
The protein should be reconstituted using sterile deionized water or the buffer specified on the lot-specific datasheet. A typical GTPase-compatible buffer contains 20 mM HEPES pH 7.4, 150 mM NaCl, and 1 mM MgCl₂. Always refer to the product datasheet for lot-specific recommendations.
How should Recombinant Human ATL1 Protein be stored to maintain stability?
For long-term storage, aliquot the protein and keep at −80°C to avoid repeated freeze-thaw cycles, which can compromise GTPase activity and protein integrity. Working stocks may be kept at 4°C for up to 48 hours. Adding a carrier protein such as 0.1% BSA is recommended when working at low protein concentrations.
What experimental applications is Recombinant Human ATL1 Protein suitable for?
Recombinant Human ATL1 Protein is suitable for GTPase activity assays, in vitro liposome membrane fusion assays, protein–protein interaction studies (pull-down, SPR, co-IP), Western blot, and ELISA. It is also used in mechanistic studies of hereditary spastic paraplegia (HSP) pathogenesis.
Why is it important to avoid repeated freeze-thaw cycles with ATL1 protein?
Repeated freeze-thaw cycles can cause protein aggregation, denaturation, and loss of GTPase enzymatic activity. To preserve protein function, aliquot the reconstituted protein into single-use volumes before freezing. This ensures each experiment uses optimally active protein.
How can I verify the quality of Recombinant Human ATL1 Protein upon receipt?
Upon receipt, perform SDS-PAGE to confirm the expected molecular weight and purity as indicated on the lot-specific datasheet. For functional validation, a GTPase activity assay using malachite green or a coupled enzyme system is recommended. Compare results to the datasheet's stated specific activity where available.
References
- Hu J, Shibata Y, Voss C, et al. Membrane proteins of the endoplasmic reticulum induce high-curvature tubules. Science. 2008;319(5867):1247-1250. doi:10.1126/science.1153634
- Orso G, Pendin D, Liu S, et al. Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature. 2009;460(7258):978-983. doi:10.1038/nature08280
- Zhao X, Alvarado D, Rainier S, et al. Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia. Nat Genet. 2001;29(3):326-331. doi:10.1038/ng758
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.
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