Ribosome Affinity Purification (RAP)

Ribosomes play an important role in protein synthesis. Abnormal synthesis of ribosomes is associated with various diseases, such as hematologic diseases and cancers. In recent years, ribosome analysis has gradually become a hotspot. As a leading service provider of ribosome purification, Creative Biolabs will provide customized ribosome affinity purification (RAP) and ribosome analysis services according to your requirements.

Introduction to RAP

Ribosomes are composed of large and small subunits, each of which is an RNA/protein complex. In the process of biological development, the morphology of ribosomes has changed, and the structural changes have also provided a new direction for the study of interacting proteins. To probe this protein interaction, the RAP method was proposed, which can identify hundreds of ribosome-associated proteins, as well as a variety of RNA and protein modifying enzymes. Currently, this technique plays an important role in ribosome analysis.

RAP is considered to be a method to rapidly purify ribosomes for further relevant information. The method relies on the expression of tagged versions of specific ribosomal proteins (RPs), and IgG-coupled spherical microbeads (matrix) efficiently recover ribosomes from cell extracts with the assistance of the tagged proteins. It is worth noting that this method plays an important role in analyzing all the information associated with the ribosome. Most importantly, RPs and their modifications can be mapped using mass spectrometry. This approach provides a promising direction for further exploration of gene expression patterns.

Fig.1 Schematic of RAP. (Halbeisen, 2009)

Ribosomes are composed of RPs and ribosomal RNA (rRNA). The key to the success of RAP is to select an RP suitable for labeling affinity purification. Furthermore, it is worth noting that fusion labels do not affect RP function. Affinity-labeled ribosomes are captured with IgG-conjugated spherical microspheres and released from the matrix with appropriate handling.

Advantages

1. RAP can quickly obtain purified ribosomes.
2. Avoid the contamination of impurities such as lipid rafts and pseudopolysomes.
3. The extraction solution of the product is free of sucrose, which is beneficial to subsequent mass spectrometry analysis.
4. RNA isolation is relatively simple, which is convenient for subsequent experiments.

Disadvantages

1. It is an overview of ribosome-related information.
2. It is not possible to distinguish whether the mRNA is bound to one or more ribosomes.
3. It does not indicate the extent to which the information has been translated.

After decades of accumulation, we have made great progress and accumulated rich experience in the field of ribosome purification and analysis. With our professional team of scientists and rich experience, Creative Biolabs is committed to providing optimal RAP service to customers around the world. Please contact us for more details.

Reference

1. Halbeisen, R. E.; et al. Affinity purification of ribosomes to access the translatome. Methods. 2009, 48(3): 306-310.