Name: how detergents solubilize membrane proteins
Uploaded: 2023-04-30T09:16:15.000Z
Duration: 1 min 18 s
Channel: JoVE

K12 Standards

Alabama Course of Study Science

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.4.a

detergent-purification-of-membrane-proteins

Detergent Purification of Membrane Proteins

Detergents contain hydrophilic head groups and hydrophobic tails, which allow them to form spherical micelles; therefore, they are widely used to purify membrane proteins.
There are three types of detergents &#8211; ionic, non-ionic, and zwitterionic.
Ionic detergents such as sodium dodecyl sulfate have charged head groups, whereas non-ionic detergents such as Triton X-100 are uncharged.
Zwitterionic detergents like CHAPS have headgroups with both positive and negative charges.
Non-ionic and zwitterionic detergents are most commonly used for protein purification as they allow the protein to retain its functional form. In contrast, ionic detergents can denature the target proteins.
Integral membrane proteins are purified in three distinct stages. First, the detergent micelle interacts with the outer bilayer of the membrane.
Next, the membrane is solubilized, with the formation of lipid-detergent and protein-detergent complexes.
Once the detergents are removed, the protein can be purified using techniques such as affinity chromatography.

detergent purification of membrane proteins

Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and it depends upon the size and structure of the detergent monomer. The formation of CMC is also affected by temperature and pH. The structure and concentration of a detergent and its micelle structure determine its ability to solubilize the lipid bilayer.
For protein purification, the protein and the detergent concentration should be higher than the CMC. There should be a minimum of one micelle for each membrane protein. The detergent: lipid ratio also plays an essential role in protein purification, and it is determined using the trial and error method for each target protein. Some proteins can be extracted with a detergent-lipid ratio ranging from 0.1:1 to 1:1, but the lipid bilayer remains undisturbed. While in ratios ranging from 1:1 to 2:1, the detergent molecules solubilize the membrane, resulting in mixed micelles with lipid, protein, and detergent. If the ratio is increased to 10:1, all the lipid molecules in the lipid-protein complex will be replaced by the detergent molecule.
The choice of detergent depends on whether the purified protein should be functionally active or inactive. Detergents with a bigger polar head or longer alkyl chain are considered milder than the detergent with a smaller polar head or shorter alkyl chain. The membrane composition also affects the choice of detergents. For example, Triton X-100 acts on the bacterial inner membrane but cannot solubilize the outer membrane. In contrast, digitonin is known to work specifically on the eukaryotic plasma membrane.

Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while ...

Education

JoVE Core

Cell Biology

Unit 1

Membrane Structure and Components

KEY TERMS AND CONCEPTS

how detergents solubilize membrane proteins

Detergents help solubilize membrane proteins so they can be purified from the lipid bilayer. Their hydrophobic region can replace membrane phospholipids and keep integral membrane proteins in solution.

A detergent works well only when its monomers reach the critical micelle concentration, or CMC. At that point, the molecules assemble into micelles, which are small clusters of detergent. Above the CMC, free detergent monomers stay in equilibrium with the micelles. The CMC varies with the detergent’s size and structure, and it is also affected by temperature and pH.

Both detergent structure and detergent amount affect how well a membrane is solubilized. For purification, the protein and detergent levels should be above the CMC, and there should be at least one micelle for each membrane protein. The detergent-to-lipid ratio is usually found by trial and error for each target protein.

Different ratios produce different results. At ratios from 0.1:1 to 1:1, some proteins can be extracted while the lipid bilayer stays intact. At ratios from 1:1 to 2:1, the membrane is solubilized and mixed micelles form with lipid, protein, and detergent. At a ratio of 10:1, the detergent can replace the lipid molecules in the lipid-protein complex.

The choice of detergent also depends on whether the purified protein should stay active or become inactive. Detergents with a larger polar head or a longer alkyl chain are considered milder than detergents with a smaller polar head or a shorter alkyl chain. Membrane type matters too. Triton X-100 acts on the bacterial inner membrane but does not solubilize the outer membrane. Digitonin, on the other hand, works specifically on the eukaryotic plasma membrane.

How Detergents Solubilize Membrane Proteins

how-detergents-solubilize-membrane-proteins

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Detergents help solubilize membrane proteins so they can be purified from the lipid bilayer. Their hydrophobic region can replace membrane phospholipids and keep integral membrane proteins in solution. A detergent works well only when its monomers reach the critical micelle concentration, or CMC. At that point, the molecules assemble into micelles, which are small clusters of detergent. Above the CMC, free detergent monomers stay in equilibrium with the micelles. The CMC varies with the...

protein diffusion in the membrane

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Membrane proteins can move across the cell surface by lateral diffusion, and they can also rotate in place. Scientists confirmed lateral movement with a cell fusion experiment. Mouse and human cells were fused to make hybrid cells. The membrane proteins on the two cells were tagged with red and green fluorescent markers. At first, each color stayed on its own side of the fused cell. After 40 minutes, the colors mixed across the whole cell surface, showing that the proteins had moved laterally. <br...

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key terms and concepts

Learn how detergents solubilize membrane proteins, form micelles at the CMC, and help choose the right detergent for purification in high school biology.

How Detergents Solubilize Membrane Proteins | JoVE K12

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How Detergents Solubilize Membrane Proteins

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