Translating Ribosome Affinity Purification (TRAP)

Creative Biolabs is the world's leading provider of ribosome analysis services. With our extensive experience in ribosome research, we are proud to offer our clients translational ribosome affinity purification (TRAP) services at the highest quality and most competitive prices.

Introduction to TRAP

In recent years, the analysis of RNA transcript levels by different techniques such as microarray and RNA-seq has contributed to the advancement of biology. However, RNA expression profiles present some limitations in studying tissues composed of multiple cell types. Therefore, TRAP has been proposed as a promising strategy that allows the isolation of transcripts from intact tissues of selected cell types without dissociation.

TRAP plays an important role in the isolation of ribosome-bound mRNA from specific cell populations from tissues. By epitope tagging of ribosomal proteins in a cell-type-specific manner, polysomes can be selectively purified without laborious cell dissociation and sorting steps. In addition, TRAP provides the closest approximation to cell-type-specific gene expression profiles in intact tissues, since gene expression does not continue after tissue lysis, and the translationome is more representative of the steady-state proteome than the transcriptome.

Fig.1 Polysome profiling using TRAP. (Goldenkova-Pavlova, 2018)

Procedures of TRAP

TRAP relies on the indirect targeting of mRNA. Indirect labeling of mRNA is achieved by adding an affinity tag such as enhanced GFP (EGFP) to the large ribosomal subunit protein L10a. The cell type of interest is targeted with the appropriate genetic elements to express the EGFP-L10a transgene. Translating polysomes from non-targeted cells are not labeled with EGFP while those from targeted cells have EGFP tags. Lysis of all cells releases both labeled and unlabeled polysomes, and only labeled polysomes are captured on an anti-GFP affinity matrix that can be used to purify cell-type-specific mRNA associated with labeled polysomes. The mRNA obtained in this way can then be analyzed in different ways, such as northern blotting, microarray, quantitative PCR (qPCR), or RNA sequencing.

Fig.2 The TRAP strategy. (Kulicke, 2014)

Advantages

It does not require fixation or dissociation of tissue to capture mRNA.

• TRAP has higher sensitivity than other methods.
• TRAP enables in situ analysis of mRNA translation profiles in whole cells.
• TRAP transgenes are labeled with EGFP for cell types of interest, allowing visualization.
• TRAP revealed that the post-translational mRNA content of cells was closer to the protein.

Creative Biolabs is committed to providing ribosome analysis services. Our services include not only ribosome extraction and isolation, but also analysis of ribosome structure and associated factors. If you have any questions about ribosome-related projects, please contact us in time.

References

1. Goldenkova-Pavlova, I. V.; et al. Affinity purification of ribosomes to access the translatome. Methods. 2018, 20(1): 33.
2. Kulicke, R.; et al. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature protocols. 2014, 9(6): 1282-1291.