Sucrose Gradient Centrifugation

Sucrose density fractionation has long been considered the gold standard for monitoring active translation. Creative Biolabs is a leading global provider of ribosome isolation and analysis services. Our expert team provides the most professional solutions and advanced technology to promote your research.

What is Sucrose Gradient Centrifugation?

Misregulation of translation can lead to a variety of diseases. The cellular mRNA translation process is inseparable from the ribosome. Actively translated mRNA is usually bound by several ribosomes and can be separated from small (40S) and large (60S) ribosomal subunits and 80S monomers by sucrose gradient centrifugation. Total RNA was isolated from different parts of the multimer gradient and detected by different methods.

Sucrose gradient centrifugation is considered a common and efficient method for characterizing subcellular particles. The technique separates targets based on a density gradient of sucrose. The main principle is that the sample rotates under centrifugal force, and the centrifugal force generated by the rotation of the sample causes the molecules to move in the medium. Different molecules move through the gradient until their density is the same as that of the surrounding medium. This allows molecules of different densities to separate at different layers, which can then be used in various recycling processes.

Fig.1 Schematic of a sucrose gradient fractionation of ribosomes. (Gerashchenko, 2017)

Applications of Sucrose Gradient Centrifugation

• This centrifugal method can be used to separate macromolecules such as DNA and RNA.
• This can also be used to analyze protein complexes, as well as to determine the size and density of other macromolecules.
• In addition, this method plays a significant role in the isolation of ribosomes as well as membrane components, centrosomes, and subcellular organelles.

Limitations of Sucrose Gradient Centrifugation

Although sucrose density fractionation plays an important role in the separation of ribosomes as well as other molecules, there are some disadvantages:

• Need special equipment, for example, it requires conditions such as ultracentrifuge, and gradient fractionation system.
• The procedure is time-consuming and does not allow multiple samples to be processed simultaneously.
• The samples will be diluted when added to the sucrose solution, therefore, more elaborate subsequent steps are required to obtain samples of sufficient quality for downstream experiments.
• The obtained samples are easily contaminated by macromolecules such as lipid rafts and pseudopolysomes.

Creative Biolabs provides customized services to global customers according to different experimental goals. We will help you design the optimal extraction protocols to achieve the best results. Please feel free to contact us.

Reference

1. Gerashchenko, M. V.; Gladyshev, V. N. Ribonuclease selection for ribosome profiling. Nucleic acids research. 2017, 45(2): e6-e6.