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.

wasmiddelzuivering van membraaneiwitten

Er worden detergenten gebruikt om de integrale eiwitten van het membraan te zuiveren. Het hydrofobe deel van het detergens kan membraanfosfolipiden vervangen terwijl de membraaneiwitten oplosbaar worden gemaakt. Wanneer detergentmonomeren een specifieke concentratie bereiken in een oplossing die kritische micelconcentratie (CMC) wordt genoemd, vormen ze micellen. Boven CMC blijft de concentratie van de detergentmonomeren in evenwicht met de micel. Het aantal detergentmonomeren dat aanwezig is in de CMC varieert voor elk detergens en hangt af van de grootte en structuur van het detergentmonomeer. De vorming van CMC wordt ook beïnvloed door temperatuur en pH. De structuur en concentratie van een detergens en zijn micelstructuur bepalen het vermogen ervan om de lipidedubbellaag oplosbaar te maken.

Voor eiwitzuivering moeten de eiwit- en detergensconcentratie hoger zijn dan de CMC. Er moet minimaal één micel voor elk membraaneiwit zijn. De verhouding wasmiddel:lipide speelt ook een essentiële rol bij de eiwitzuivering en wordt voor elk doeleiwit bepaald met behulp van de 'trial and error'-methode. Sommige eiwitten kunnen worden geëxtraheerd met een wasmiddel-lipideverhouding variërend van 0,1:1 tot 1:1, maar de lipidedubbellaag blijft ongestoord. In verhoudingen variërend van 1:1 tot 2:1 lossen de wasmiddelmoleculen het membraan op, wat resulteert in gemengde micellen met lipide, eiwit en wasmiddel. Als de verhouding wordt verhoogd tot 10:1, zullen alle lipidemoleculen in het lipide-eiwitcomplex worden vervangen door het wasmiddelmolecuul.

De keuze van het wasmiddel hangt af van de vraag of het gezuiverde eiwit functioneel actief of inactief moet zijn. Wasmiddelen met een grotere polaire kop of een langere alkylketen worden als milder beschouwd dan wasmiddelen met een kleinere polaire kop of een kortere alkylketen. De membraansamenstelling heeft ook invloed op de keuze van wasmiddelen. Triton X-100 werkt bijvoorbeeld in op het bacteriële binnenmembraan, maar kan het buitenmembraan niet oplosbaar maken. Het is daarentegen bekend dat digitonine specifiek op het eukaryotische plasmamembraan werkt.

Wasmiddelzuivering van membraaneiwitten

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

Celbiologie

Unit 1

Eenheid 1

Membrane Structure and Components

Membraanstructuur en componenten

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

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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detergent purification of 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...

protein diffusion in the membrane

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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

cell biology

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9-12.B.1.2.a

How Detergents Solubilize Membrane Proteins

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