Rabbit Reticulocyte Lysate (RRL) Cell-Free System Service

The Rabbit Reticulocyte Lysate (RRL) Cell-Free Protein Synthesis (CFPS) System is the classic and most sensitive eukaryotic in vitro translation system. Derived from rabbit reticulocytes (immature red blood cells), the lysate is naturally rich in the translational machinery—specifically high concentrations of ribosomes, tRNAs, and initiation/elongation factors—required for efficient hemoglobin synthesis . After treatment to inactivate endogenous mRNA (typically with micrococcal nuclease), RRL becomes an open, highly active system primed for translating exogenous mRNA or DNA templates.

CD Biosynsis offers a premier RRL CFPS Service tailored for sensitive biochemical assays, high-fidelity translation, and PTM studies. RRL is the preferred choice when low background, high sensitivity, and authentic eukaryotic translation initiation are paramount. Its superior fidelity makes it essential for investigating regulatory elements of eukaryotic translation, such as IRES structures, signal peptide processing, and phosphorylation-dependent regulation , offering results that closely mimic the in vivo environment.

Highlights

Key advantages of utilizing the RRL CFPS System:

• Highest Translation Fidelity: RRL initiation factors closely resemble those found in human cells, providing the most accurate model for eukaryotic translation mechanism research .
• High Sensitivity and Low Background: Minimal endogenous protein synthesis allows for extremely sensitive detection methods, crucial for detecting low-abundance regulatory proteins.
• PTM Compatibility: Easily supplemented with microsomes to enable accurate processing of secretory and membrane proteins, including signal peptide cleavage and N-linked glycosylation .
• Robust for Toxin/Regulatory Proteins: Ideal for expressing proteins that regulate cell growth or are highly toxic to living cells, such as kinases and transcription factors.

Applications

RRL is critical for highly specific research in biochemistry and molecular biology:

Eukaryotic Translation Regulation

Studying the activity of IRES elements, upstream open reading frames (uORFs), and cap-dependent/independent initiation mechanisms.

Protein Interaction and Folding Assays

Rapid synthesis of protein probes for co-immunoprecipitation, binding studies, and chaperone-assisted folding analysis.

In Vitro Mutagenesis Screening

Quickly translating and analyzing small batches of site-directed mutants for changes in activity or translation efficiency.

Radioactive Labeling and Detection

Optimal platform for highly sensitive detection using [35S]methionine or [14C]leucine due to its low background.

Key Features & Sensitivity

Why RRL is the standard for high-fidelity eukaryotic translation:

High Initiation Factor Concentration

Reticulocytes are specialized for rapid hemoglobin synthesis, ensuring a high natural concentration of critical eukaryotic initiation factors (eIFs).

Micrococcal Nuclease Treatment

Eliminates endogenous mRNA, ensuring that protein synthesis is entirely dependent on the exogenous template provided, reducing background noise.

Capped mRNA Preference

RRL strongly prefers 5'-capped mRNA templates, faithfully replicating the cap-dependent initiation mechanism prevalent in eukaryotes.

Microsome Supplementation

We offer RRL reactions supplemented with ER microsomes to enable translocation, signal peptide cleavage, and N-linked glycosylation of membrane/secreted proteins.

Compatibility with Labeling

Ideal for labeling with non-radioactive markers, fluorescent probes, or low-concentration stable isotopes for sensitive structural studies.

Workflow

Our systematic approach for high-fidelity RRL CFPS protein synthesis:

• Template Preparation: Gene is supplied, and we prepare high-quality cDNA or optimized capped mRNA template.
• System Setup: The template is combined with the high-activity RRL lysate, energy mix, and any required PTM supplements (e.g., microsomes).
• In Vitro Synthesis: The reaction is incubated at 30°C for short durations (1-2 hours) for maximum translation fidelity and yield.
• Assay/Detection: Due to the high sensitivity of the system, the product is often used directly for functional assays or detected via autoradiography (if radioactive labels are used).
• Purification and Analysis (Optional): Purification is performed for larger-scale needs, followed by SDS-PAGE and Western blot analysis.
• Delivery: Delivery of synthesized protein or detailed translation reaction data (e.g., band intensity analysis).

We provide essential assurance for high-quality RRL expression outcomes:

• Confirmed mRNA Integrity: Ensures the mRNA template is of high quality and correctly capped for optimal RRL translation.
• Optimized PTM Conditions: Precise control over the oxidative environment and microsome concentration for targeted processing and disulfide bond formation.
• High Yield and Fidelity: Guaranteed performance metrics for translation initiation efficiency compared to established standards.
• Technical Support: Consultation on experimental design for specific translation mechanism studies (e.g., frameshifting, stop-codon readthrough).