RiboLace Pro

Mechanism of active ribosome capture in Ribo-seq workflows

RiboLace Pro exploits the interaction between a puromycin analog (3P) and the ribosomal A-site to selectively capture actively translating ribosomes. Puromycin mimics the 3′ end of aminoacyl-tRNA and can enter the catalytic center of ribosomes engaged in peptide elongation. In RiboLace Pro, the molecule is immobilized on magnetic beads, allowing translation-competent ribosomal complexes to be isolated through affinity capture.

Because the interaction relies on puromycin-like chemistry associated with the catalytic center of active ribosomes, the enrichment step preferentially isolates ribosomes engaged in translation relative to inactive or dissociated complexes. Following nuclease digestion, these ribosomes remain associated with short RNA fragments that correspond to ribosome-protected regions of transcripts.

Magnetic separation of the captured complexes therefore enriches ribosome-protected fragments derived from actively translated mRNA regions. These fragments can then be purified and processed using standard Ribo-seq library preparation workflows to measure ribosome occupancy and translation dynamics across the transcriptome.

Advantages over sucrose gradients and polysome profiling

Many ribosome profiling workflows use sucrose gradient fractionation or polysome profiling to separate translating ribosomes as part of upstream enrichment. These approaches rely on ultracentrifugation and fraction collection, which can add complexity and require access to an ultracentrifuge.

RiboLace Pro simplifies this step by enriching translating ribosomes directly from cell lysates using an affinity-based pull-down. Because the enrichment occurs without gradient separation, the workflow can be performed without fractionation or ultracentrifugation while still producing material suitable for downstream Ribo-seq analysis.

Library preparation and QC tools for Ribo-seq workflows

RiboLace Pro enrichment workflows integrate with standard small RNA-style library preparation approaches used for ribosome profiling. Compatibility with the NEXTFLEX Small RNA-Seq Kit is demonstrated in our application note, providing a clear path from enriched ribosome footprints to sequencing-ready libraries using a workflow optimized for short RNA inserts.

Figure 1. Metaprofile plot showing P-site positions for all transcripts around translation initiation sites (0 to +100 nt) and translation termination sites (0 to −100 nt). RPFs were isolated using RiboLace® Pro, and sequencing libraries were prepared using the NEXTFLEX Small RNA-Seq Kit.

Additional tools within the NEXTFLEX ecosystem can support library quality and experimental consistency. miND™ spike-in controls can be incorporated to monitor technical variation and assist with normalization across samples. Custom small RNA blockers can be used as an optional tool to suppress abundant non-target short fragments when they impact usable sequencing depth.