L-Leucine-¹³C₆ FAQs: Isotope Labeling & SILAC Use

L-Leucine-¹³C₆ is a fully ¹³C-labeled stable isotope form of the essential amino acid L-leucine, widely used as a heavy internal standard in SILAC-based quantitative proteomics and metabolic flux studies.

Product SKU

L471812

Chemical Name

L-Leucine-¹³C₆ (all six carbons replaced with ¹³C)

Isotopic Enrichment

≥99 atom % ¹³C (refer to certificate of analysis; verify with supplier)

Primary Application

SILAC-based quantitative proteomics; stable isotope tracer studies

Mass Shift vs. Unlabeled Leucine

+6 Da per leucine residue (six ¹³C substitutions)

Reference Comparator

Sigma-Aldrich No. 605239 (analogous product; verify specifications)

Storage Recommendation

Store at 2–8 °C, protected from moisture and light (verify CoA for exact conditions)

What Is L-Leucine-¹³C₆ and How Is It Made?

L-Leucine-¹³C₆ is a stable, non-radioactive isotopologue of the branched-chain amino acid L-leucine in which all six carbon atoms are replaced with the stable heavy carbon isotope ¹³C. Because ¹³C is stable (not radioactive), this compound poses no radiation hazard and can be handled under standard laboratory conditions.

The compound is typically produced by biosynthetic or chemical synthesis routes starting from ¹³C-enriched carbon sources, followed by high-performance purification to achieve isotopic enrichment typically ≥99 atom % ¹³C. The resulting molecule is chemically and biochemically identical to natural L-leucine but is distinguished by its increased molecular mass (+6 Da relative to the unlabeled form), which is readily resolved by modern mass spectrometers.

Note: Exact synthesis route and certified enrichment values should be confirmed on the product Certificate of Analysis (CoA).

Key Applications: SILAC and Quantitative Proteomics

The most established application of L-Leucine-¹³C₆ is in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). In a typical SILAC experiment, one cell population is cultured in medium containing the heavy labeled amino acid (e.g., L-Leucine-¹³C₆) while a control population is grown in medium containing the normal (light) L-leucine. After several cell doublings, the heavy label is incorporated into virtually all newly synthesized proteins. The two cell populations are then combined, digested with a protease (commonly trypsin), and analyzed by LC-MS/MS.

Because leucine does not appear at the C-terminus of tryptic peptides (trypsin cleaves after Lys and Arg), it is incorporated into internal peptide positions, providing a +6 Da mass shift per residue. This mass difference allows precise relative quantification of protein abundance between experimental conditions directly from MS1 spectra.

Additional applications include:

• Metabolic flux analysis and tracer studies
• Biomarker discovery and validation
• Drug metabolism and pharmacokinetic studies using isotope dilution mass spectrometry (IDMS)
• In vitro translation and cell-free protein synthesis studies

Handling, Storage, and Quality Considerations

Storage: L-Leucine-¹³C₆ should generally be stored at 2–8 °C in a tightly sealed container, protected from moisture and light. Always refer to the product-specific Certificate of Analysis (CoA) for confirmed storage conditions, as recommendations may vary by lot and formulation.

Solubility: Like unlabeled L-leucine, L-Leucine-¹³C₆ is soluble in water and dilute acid or base, but has limited solubility in most organic solvents. For SILAC media preparation, dissolve in sterile water or cell culture-grade balanced salt solution and filter-sterilize before adding to leucine-free medium.

Isotopic purity: High isotopic purity (≥99 atom % ¹³C) is critical for SILAC experiments to minimize spectral overlap between heavy and light peptide pairs. Always request and review the CoA for isotopic enrichment data.

Chemical purity: For quantitative applications, chemical purity should also be high. Verify the purity specification on the CoA before use.

Handling precautions: No special radiation precautions are required. Standard laboratory PPE (gloves, lab coat, eye protection) is recommended. Refer to the Safety Data Sheet (SDS) for full hazard information.

Ordering L-Leucine-¹³C₆ from Aladdin Scientific

Aladdin Scientific supplies L-Leucine-¹³C₆ under SKU L471812. This product is analogous to Sigma-Aldrich catalog number 605239 and is suitable for SILAC proteomics and stable isotope tracer research.

When placing an order, please consider:

• Quantity requirements: Estimate your total experimental consumption before ordering to avoid freeze-thaw cycling of unused material.
• Certificate of Analysis: A lot-specific CoA is available upon request and should be reviewed prior to use to confirm isotopic enrichment, chemical purity, and storage conditions.
• Shipping and storage: The product ships with appropriate cold-chain packaging. Inspect upon receipt and store immediately under recommended conditions.
• Custom quantities or formulations: Contact Aladdin Scientific customer service for bulk pricing or custom packaging inquiries.

For technical questions about this product, please contact our technical support team through the Aladdin Scientific website.

Frequently asked questions

What does the ¹³C₆ designation mean for L-Leucine-¹³C₆?

The ¹³C₆ designation indicates that all six carbon atoms in the L-leucine molecule have been replaced with the stable heavy isotope ¹³C (carbon-13). This results in a molecule that is chemically identical to natural L-leucine but has a molecular mass approximately 6 Da heavier, making it distinguishable by mass spectrometry without the use of radioactivity.

Is L-Leucine-¹³C₆ radioactive, and what safety precautions are needed?

No, L-Leucine-¹³C₆ is not radioactive. ¹³C is a stable, non-radioactive isotope of carbon, so no radiation permits, shielding, or special disposal procedures for radioactive materials are required. Standard laboratory precautions apply: wear gloves, a lab coat, and eye protection, and consult the Safety Data Sheet (SDS) for complete hazard information.

How many cell doublings are needed for full SILAC incorporation of L-Leucine-¹³C₆?

In a standard SILAC protocol, cells are typically cultured in heavy-amino-acid-containing medium for at least five to six cell doublings to achieve >95% isotopic incorporation into the cellular proteome. The exact number may vary depending on cell type and growth rate. It is strongly recommended to verify incorporation efficiency by MS analysis of a test sample before proceeding with quantitative experiments.

How does L-Leucine-¹³C₆ compare to L-Leucine-d₁₀ (deuterium-labeled leucine) for SILAC?

L-Leucine-¹³C₆ (¹³C-labeled) is generally preferred over deuterium-labeled leucine for SILAC because ¹³C substitution does not significantly alter chromatographic retention time relative to the light form, whereas deuterium-labeled compounds can exhibit slight retention time shifts in reversed-phase LC, complicating data analysis. The +6 Da mass shift provided by ¹³C₆ also offers excellent separation from the light isotopologue in MS1 spectra.

What is the recommended concentration of L-Leucine-¹³C₆ in SILAC cell culture medium?

The concentration of L-Leucine-¹³C₆ in SILAC medium should match the leucine concentration used in the standard cell culture medium formulation for the cell line being studied (for example, DMEM typically contains approximately 105 mg/L L-leucine). Leucine-free basal medium is supplemented with the heavy leucine at an equivalent molar concentration to the standard formulation. Always consult the specific SILAC protocol and the basal medium formulation for your cell type.

How should I verify isotopic incorporation efficiency in my SILAC experiment?

To verify incorporation efficiency, collect a small aliquot of heavy-labeled cells after the recommended number of doublings, lyse and digest the proteins with trypsin, and analyze the resulting peptides by LC-MS/MS. Calculate the ratio of heavy to light peptide signal for leucine-containing peptides; incorporation efficiency is typically expressed as a percentage of the heavy isotopologue signal relative to the total signal. An incorporation rate of ≥95% is generally considered acceptable for quantitative SILAC experiments.

References

1. Ong, S.E. et al. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & Cellular Proteomics, 1(5), 376–386. doi:10.1074/mcp.M200025-MCP200
2. Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews Molecular Cell Biology, 7(12), 952–958. doi:10.1038/nrm2067
3. Sigma-Aldrich. L-Leucine-¹³C₆, catalog number 605239. Product information page.

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