Difference Between Sucrose Gradient and Sucrose Cushion Ultracentrifugation

The key difference between sucrose gradient and sucrose cushion ultracentrifugation is that in sucrose gradient ultracentrifugation, a continuous sucrose gradient is used, while in sucrose cushion ultracentrifugation, a discontinuous sucrose gradient is used.

Sucrose gradient and sucrose cushion ultracentrifugation are two similar types of techniques used to separate specific types of macromolecules. In both techniques, a sucrose density gradient is used. But in sucrose gradient ultracentrifugation, a continuous density gradient is used while, in sucrose cushion ultracentrifugation, a discontinuous density gradient is used.

CONTENTS

1. Overview and Key Difference
2. What is Sucrose Gradient Ultracentrifugation
3. What is Sucrose Cushion Ultracentrifugation
4. Similarities Between Sucrose Gradient and Sucrose Cushion Ultracentrifugation
5. Side by Side Comparison – Sucrose Gradient vs Sucrose Cushion Ultracentrifugation in Tabular Form
6. Summary

What is Sucrose Gradient Ultracentrifugation?

Sucrose gradient ultracentrifugation is a technique that can be used to fractionate macromolecules such as DNA, RNA and proteins. There are several steps in this technique. They are sucrose gradient preparation, centrifugation, separation and elution. Gradient preparation is the key step in this technique. In this technique, the sample containing different size macromolecules is layered on the surface of a sucrose gradient column. When centrifuging, different sized macromolecules sediment through the sucrose gradient column at different rates. Sedimentation depends on several factors, including the centrifugation force, size shape and the density of macromolecules, density and viscosity of the gradient. At the end of the centrifugation, macromolecules are spatially separated by density. Larger macromolecules (high-density macromolecules) sediment towards the bottom. Lighter ones (low-density macromolecules) remain at the top of the gradient. Therefore, the molecules separate as different bands. Then the bands should be separated, and the purification of the specific macromolecule from sucrose has to be done.

On the application side, this method is extensively used for fractionation of DNA molecules. Sucrose gradient ultracentrifugation is commonly used to characterize protein complex size and composition. Moreover, this technique is used for the partial purification of mRNA.

What is Sucrose Cushion Ultracentrifugation?

Sucrose cushion ultracentrifugation is another technique that enables the fractionation of macromolecules. Unlike sucrose density gradient ultracentrifugation, sucrose cushion ultracentrifugation uses a discontinuous density gradient. The sucrose concentration increases from top to bottom in discrete steps. In this technique, separation is achieved by placing the clarified extract on top of a small volume of a sucrose solution in a centrifuge tube, the so-called sucrose cushion. This allows better separation in high-speed centrifugation. When the molecules separate as a band between the sucrose cushion, the white band can be collected and purified. Moreover, impurities can be further removed using sucrose cushion method. Sucrose cushion method allows 60-70% of the tube to be filled with the sample. Hence, the sucrose cushion method enables a greater volume of sample to be processed.

What are the Similarities Between Sucrose Gradient and Sucrose Cushion Ultracentrifugation?

• Sucrose gradient and sucrose cushion ultracentrifugation are two techniques that are used to separate a mixture of macromolecules.
• In both methods, a sucrose gradient is used.
• Centrifugation is a step in both methods.
• The molecules sediment as a band or zone in each method.

What is the Difference Between Sucrose Gradient and Sucrose Cushion Ultracentrifugation?

Sucrose gradient ultracentrifugation is a technique that uses a continuous sucrose gradient in the centrifuge tube while sucrose cushion ultracentrifugation is a technique that uses a discontinuous sucrose gradient. Thus, this is the key difference between sucrose gradient and sucrose cushion ultracentrifugation.

Moreover, sucrose cushion ultracentrifugation allows a greater volume of the sample to be processed while sucrose gradient ultracentrifugation allows a comparatively low volume of the sample to be processed.

Below infographic tabulates more differences between sucrose gradient and sucrose cushion ultracentrifugation.

Summary – Sucrose Gradient vs Sucrose Cushion Ultracentrifugation

Sucrose gradient ultracentrifugation separates macromolecules using a continuous sucrose gradient. In contrast, sucrose cushion ultracentrifugation uses a discontinuous sucrose gradient in order to separate specific type of particles in a mixture. So, this is the key difference between sucrose gradient and sucrose cushion ultracentrifugation. The sucrose cushion method allows the collection of morphologically intact particles since it does not cause mechanical stress, unlike sucrose gradient ultracentrifugation, which crushes molecules to the bottom of the tube. Moreover, sucrose cushion ultracentrifugation allows a greater volume of the sample to be processed than the sucrose gradient ultracentrifugation.

Reference:

1. Dijkstra, Jeanne, and Cees P. de Jager. “Density-Gradient Centrifugation.” SpringerLink, Springer, Berlin, Heidelberg, 1 Jan. 1998, Available here.

Image Courtesy:

1. “Density gradient”- Mlw3559 at English Wikibooks. – Transferred from en.wikibooks to Commons. (CC BY-SA 2.5) via Commons Wikimedia