Recombinant Human LAMTOR5 Protein: Handling & Usage Protocol

Recombinant Human LAMTOR5 Protein is a research-grade recombinant protein representing the late endosomal/lysosomal adaptor and MAPK and mTOR activator subunit 5, supplied by Aladdin Scientific for use in mTORC1 pathway studies, protein interaction assays, and lysosomal signaling research.

Product Name

Recombinant Human LAMTOR5 Protein

SKU

rp370374

Protein Target

LAMTOR5 (Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 5)

Species Origin

Human

Primary Research Application

mTORC1 signaling, Ragulator complex studies, lysosomal biology, protein–protein interaction assays

Supplier

Aladdin Scientific

Background: LAMTOR5 and the Ragulator Complex

LAMTOR5, also known as Hepatitis B X-interacting protein (HBXIP) or Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 5, is a member of the Ragulator complex—a pentameric protein assembly anchored to the lysosomal surface. The Ragulator complex (comprising LAMTOR1–5) acts as a scaffold that recruits the Rag GTPases and the mechanistic target of rapamycin complex 1 (mTORC1) to the lysosome, a critical step for amino-acid-dependent mTORC1 activation.

Beyond its role in mTORC1 regulation, LAMTOR5 has been implicated in MAPK signaling, cell proliferation, and, through its original characterization as HBXIP, in interactions with hepatitis B virus X protein. Its dual functions in oncogenic signaling and lysosomal nutrient sensing make it a relevant research target in cancer biology and metabolic research.

Reconstitution and Storage Guidelines

Reconstitution: Upon receipt, briefly centrifuge the vial before opening to collect any lyophilized material from the cap and sides. Reconstitute the protein in sterile, ultrapure water or an appropriate buffer (e.g., phosphate-buffered saline, PBS, pH 7.4) to the desired working concentration. Allow the solution to equilibrate for 10–15 minutes at room temperature with gentle mixing; avoid vigorous vortexing to prevent protein denaturation.

Short-term storage: Reconstituted protein should be stored at 4 °C for up to 1 week when frequent access is required.

Long-term storage: For extended storage, aliquot the reconstituted protein into single-use volumes to minimize freeze-thaw cycles and store at –20 °C or –80 °C. Addition of a carrier protein (e.g., 0.1% BSA) or glycerol (to a final concentration of 10–20%) may improve stability during long-term storage.

Important: Avoid repeated freeze-thaw cycles, as these can lead to protein aggregation and loss of biological activity. Specific reconstitution concentrations, buffer compositions, and storage conditions recommended by Aladdin Scientific should be confirmed on the product datasheet for SKU rp370374.

Experimental Applications and Usage Protocol

Recombinant Human LAMTOR5 Protein is suitable for a range of in vitro biochemical and cell biology applications. The following general guidance applies:

• Protein–Protein Interaction Assays (e.g., Co-IP, pull-down): LAMTOR5 can be used to study its interactions with other Ragulator subunits (LAMTOR1–4), Rag GTPases, or HBx protein. Optimize protein concentrations and buffer conditions empirically. Typical working concentrations range from 0.1 to 10 µg per reaction, but should be determined by titration.
• Structural and Biophysical Studies: Recombinant LAMTOR5 is suitable for use in techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or analytical size-exclusion chromatography to characterize binding kinetics and complex formation.
• Antibody Validation and Western Blot Standards: The recombinant protein may serve as a positive control or standard in immunoassays, including ELISA and Western blot, to validate LAMTOR5-specific antibodies.
• In Vitro Functional Reconstitution: LAMTOR5 may be incorporated into reconstituted Ragulator complexes for biochemical characterization of mTORC1 activation mechanisms.

Always include appropriate negative controls and optimize working concentrations for each specific assay format.

Quality Considerations and Troubleshooting

Purity and Identity Verification: Before use in sensitive assays, confirm protein identity and purity by SDS-PAGE or Western blot using a validated anti-LAMTOR5 antibody. Expected purity and molecular weight information should be referenced from the official Aladdin Scientific product datasheet for rp370374.

Protein Aggregation: If aggregation is observed (visible precipitation or anomalous SEC elution profile), briefly centrifuge at 10,000 × g for 5 minutes at 4 °C and use only the supernatant. Optimize buffer conditions (ionic strength, pH, detergent) to improve solubility.

Loss of Activity: If reduced biological activity is observed, verify that the protein has not undergone excessive freeze-thaw cycles. Freshly reconstituted protein generally performs best. Ensure that reducing agents (e.g., DTT or β-mercaptoethanol) are present if required for protein stability.

Endotoxin Levels: For cell-based assays, endotoxin contamination can confound results. Confirm that endotoxin levels meet the requirements of your specific assay; consult the product datasheet for endotoxin specifications.

Frequently asked questions

What is LAMTOR5 and why is it important in research?

LAMTOR5 is a subunit of the Ragulator pentameric complex anchored to the lysosomal membrane. It plays a key role in the amino-acid-dependent activation of mTORC1, a central regulator of cell growth, autophagy, and metabolism. LAMTOR5 is also implicated in MAPK signaling and, as HBXIP, in hepatitis B virus biology and cancer, making it a multifaceted research target.

How should I reconstitute Recombinant Human LAMTOR5 Protein (rp370374)?

Briefly centrifuge the vial before opening, then reconstitute the lyophilized protein in sterile ultrapure water or PBS (pH 7.4) to the desired concentration. Mix gently for 10–15 minutes at room temperature. Refer to the Aladdin Scientific product datasheet for rp370374 for the exact recommended reconstitution volume and buffer.

How do I store Recombinant Human LAMTOR5 Protein to maintain its stability?

For short-term storage (up to one week), keep the reconstituted protein at 4 °C. For long-term storage, aliquot into single-use volumes and store at –20 °C or –80 °C. Avoid repeated freeze-thaw cycles, and consider adding a carrier protein such as 0.1% BSA or 10–20% glycerol to improve long-term stability.

What assays is Recombinant Human LAMTOR5 Protein suitable for?

Recombinant Human LAMTOR5 Protein (rp370374) is suitable for protein–protein interaction assays (co-immunoprecipitation, pull-down), biophysical characterization (SPR, ITC), antibody validation (Western blot, ELISA), and in vitro reconstitution of the Ragulator complex for mTORC1 activation studies.

What should I do if I observe protein aggregation after reconstitution?

If aggregation occurs, centrifuge the reconstituted protein at 10,000 × g for 5 minutes at 4 °C and use only the clarified supernatant. Try optimizing buffer conditions such as pH, ionic strength, or the addition of mild detergent. Ensure the protein was not exposed to repeated freeze-thaw cycles prior to use.

References

1. Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM. Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1. Cell. 2012;150(6):1196-1208. doi:10.1016/j.cell.2012.07.032
2. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010;141(2):290-303. doi:10.1016/j.cell.2010.02.024
3. Meng X, et al. HBXIP as an oncoprotein in cancer. Cancer Letters. 2018. (Review reference - specific volume and pages require verification)

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