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

세제는 친수성 헤드 그룹과 소수성 테일을 함유하고 있어 구형 미셀을 형성할 수 있습니다. 따라서 멤브레인 단백질을 정제하는 데 널리 사용됩니다.
세제에는 ionic, non-ionic 및 zwitterionic의 세 가지 유형이 있습니다.
소듐 도데실 설페이트와 같은 이온 성 세제는 헤드 그룹을 충전한 반면 Triton X-100과 같은 비이온 세제는 충전되지 않았습니다.
CHAPS와 같은 Zwitterionic 세제에는 양전하와 음전하를 모두 가진 헤드그룹이 있습니다.
비이온성 및 양쪽성 이온 세제는 단백질이 기능적 형태를 유지할 수 있도록 하기 때문에 단백질 정제에 가장 일반적으로 사용됩니다. 대조적으로, 이온 성 세제는 표적 단백질을 변성시킬 수 있습니다.
일체형 멤브레인 단백질은 세 가지 뚜렷한 단계로 정제됩니다. 첫째, 세제 미셀은 멤브레인의 외부 이중층과 상호 작용합니다.
다음으로, 멤브레인은 지질 세제 및 단백질 세제 복합체의 형성과 함께 용해됩니다.
세제가 제거되면 친화성 크로마토그래피와 같은 기술을 사용하여 단백질을 정제할 수 있습니다.

막 단백질의 세제 정제

세제는 막의 필수 단백질을 정제하는 데 사용됩니다. 세제의 소수성 부분은 막 단백질을 용해시키면서 막 인지질을 대체할 수 있습니다. 세제 단량체가 임계 미셀 농도(CMC)라는 용액에서 특정 농도에 도달하면 미셀을 형성합니다. CMC 이상에서는 세제 단량체의 농도가 미셀과 평형을 유지합니다. CMC에 존재하는 세제 모노머의 수는 각 세제마다 다르며, 세제 모노머의 크기와 구조에 따라 달라집니다. CMC의 형성은 온도와 pH의 영향도 받습니다. 세제의 구조와 농도, 미셀 구조에 따라 지질 이중층을 용해시키는 능력이 결정됩니다.

단백질 정제를 위해서는 단백질과 세제 농도가 CMC보다 높아야 합니다. 각 막 단백질에는 최소 하나의 미셀이 있어야 합니다. 세제-지질 비율 역시 단백질 정제에 필수적인 역할을 하며, 각 표적 단백질에 대해 시행착오 방법을 사용하여 결정됩니다. 일부 단백질은 0.1:1~1:1 범위의 세제-지질 비율로 추출할 수 있지만 지질 이중층은 그대로 유지됩니다. 1:1 ~ 2:1 비율의 세제 분자는 막을 용해시켜 지질, 단백질 및 세제와 혼합된 미셀을 생성합니다. 비율이 10:1로 증가하면 지질-단백질 복합체의 모든 지질 분자가 세제 분자로 대체됩니다.

세제의 선택은 정제된 단백질이 기능적으로 활성이어야 하는지, 아니면 비활성이어야 하는지에 따라 달라집니다. 극성 머리가 더 크거나 알킬 사슬이 더 긴 세제는 극성 머리가 더 작거나 알킬 사슬이 더 짧은 세제보다 순한 것으로 간주됩니다. 막 구성도 세제 선택에 영향을 미칩니다. 예를 들어 Triton X-100은 박테리아 내막에 작용하지만 외막을 용해시킬 수는 없습니다. 대조적으로, 디지토닌은 진핵생물의 원형질막에 특이적으로 작용하는 것으로 알려져 있습니다.

세제는 멤브레인의 필수 단백질을 정제하는 데 사용됩니다. 세제의 소수성 부분은 멤브레인 인지질을 대체할 수 있으며 ...

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Membrane Structure and Components

막 구조 및 구성 성분

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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

what are membranes?

Membrane Receptors and Cell Signals

The plasma membrane sets the outer boundary of a cell and helps control how the cell interacts with its environment. It lets some substances enter, keeps others out, and releases some materials in controlled amounts. The membrane also needs to stay flexible so cells such as red and white blood cells can change shape as they move through narrow capillaries. The membrane surface also carries markers that help cells recognize one another. This recognition is important during early development,...

membrane fluidity

How Cell Membranes Stay Flexible

Cell membranes stay flexible because of the fluid mosaic model. The plasma membrane is a mosaic of phospholipids, cholesterol, proteins, and carbohydrates. These parts are separate but loosely attached, so the membrane keeps a fluid character. The membrane is still fairly rigid, but it can flow and self-seal after a very fine needle passes through it. Phospholipid tails also affect membrane fluidity. Saturated fatty acids in phospholipids have straight tails because they are packed with...

fluid mosaic model

Fluid Mosaic Model of the Membrane

The fluid mosaic model explains the structure and function of the plasma membrane. Scientists first identified the plasma membrane in the 1890s, and by 1915 they had identified its main chemical parts: lipids and proteins. Early electron micrographs showed a railroad track pattern, which helped lead to the first widely accepted membrane model in 1935.

membrane lipids

Membrane Lipids in Cells and Tissues

Membrane lipids give biological membranes their structure and help set their properties. In mammalian cells, lipids make up about 50% of the membrane on average. The amount can be much lower in the inner mitochondrial membrane or much higher in the myelin sheath around nerve cells. Several phospholipids are common in mammalian membranes. These include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. At physiological pH, phosphatidylserine carries a negative...

asymmetric lipid bilayer

Membrane Lipid Unevenness

Biological membranes have an uneven mix of lipids between the inner and outer layers. This membrane asymmetry gives each side a different chemical makeup and supports different cell functions. Evidence for this uneven layout came from treating an erythrocyte membrane with phospholipase. Phospholipase is an enzyme that breaks lipids into fatty acids and hydrophilic groups. It acted only on the outer layer, while the inner layer stayed intact. The treatment degraded about 80% of total membrane...

membrane asymmetry regulating transporters

membrane-asymmetry-regulating-transporters

Membrane Lipid Flipping and Transport

Membrane lipid flipping and transport help cells keep an uneven, or asymmetric, distribution of phospholipids across the membrane. This balance is controlled by flippase, floppase, and scramblase enzymes, which move lipids between the two membrane leaflets. Flippases move specific phospholipids from the outer leaflet to the inner leaflet. In eukaryotes, these enzymes are type-IV P-type ATPases, also called P4-ATPases. They are membrane pumps that use ATP and contain one transmembrane domain...

membrane carbohydrates

Membrane Sugars and Cell Identity

Membrane carbohydrates help give cells their surface identity and support plasma membrane function. The plasma membrane is a dynamic barrier made of lipids, proteins, and carbohydrates. These sugar chains have unique structural and chemical traits that help the membrane work properly for cell growth and survival. Membrane carbohydrates do not have a hydrophobic region, so they are found only on the outer surface of the cell. Sugar addition, or glycosylation, of proteins occurs in the lumens of...

membrane proteins

Facilitated Transport Across Cell Membranes

Facilitated transport uses membrane proteins to help materials cross the plasma membrane. These integral transmembrane proteins act as channels or carriers. They move selected substances through the membrane more easily than the lipid layer alone would allow. Channel proteins provide a hydrophilic, or water-friendly, path through the membrane. Their core forms a hydrated opening that lets solutes pass between the inside and outside fluids of the cell. This helps polar compounds avoid the...

lipids as anchors

Lipid Anchors in Membrane Proteins

Lipid anchors help attach proteins to the plasma membrane. In eukaryotic cells, the main lipid anchors are prenyl groups, fatty acyl groups, and glycosylphosphatidylinositol, or GPI, groups. Prenyl and fatty acyl groups anchor proteins on the cytosolic side of the membrane. GPI anchors proteins on the extracellular side. Prenylated proteins often have a CaaX motif at the carboxy terminus. In this sequence, C means cysteine, a means any aliphatic amino acid, and X means any other amino acid...

single-pass transmembrane proteins

How Membrane Proteins Span Cell Membranes

Membrane proteins help cells communicate, send signals, stick to other cells, and move molecules across the membrane. Integral membrane proteins are tightly linked to the cell membrane and carry out these jobs. Some integral membrane proteins sit only in one membrane layer. Fatty acid amide hydrolase is one example, and it is found on the cytoplasmic side of the membrane monolayer. Other integral membrane proteins are transmembrane proteins, which span across the membrane. These proteins have...

multi-pass transmembrane proteins and &#946;-barrels

multi-pass-transmembrane-proteins-and-barrels

Membrane-Spanning Helices and β-Barrels

Membrane-spanning proteins can cross the cell membrane more than once. Their transmembrane chain usually folds into either an alpha-helix or a beta-strand. Alpha-helix multi-pass proteins are common, while beta-strand proteins are found mainly in gram-negative bacteria, mitochondria, and chloroplasts. Some multi-pass alpha-helix proteins are G-protein-linked receptors, or GPCRs, and bacteriorhodopsin. GPCRs have seven transmembrane alpha-helices. Each receptor also has its own extracellular...

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

How Membrane Proteins Move Side to Side

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...

membrane domains

Lipid Rafts and Sperm Membrane Regions

Membrane domains organize specific lipids and proteins in defined parts of a cell membrane. This arrangement supports cell signaling, adhesion, and other important cellular processes. These domains can vary in size, composition, function, and how long they last. Some membrane domains are built from proteins. The membrane can contain distinct protein groups that carry out a cell’s specific functions. A clear example is the plasma membrane of a human sperm cell, which has different proteins in...

mechanisms of membrane domain formation

How Cell Membranes Form Domains

Cell membranes form domains when lipids and proteins gather into distinct regions. These regions, or membrane domains, are shaped by different physical properties that help some molecules stay together while others move apart. Some domains form through protein-protein interactions. Others depend on specific lipids, such as sphingolipids and sterols. Large proteins, including bacteriorhodopsin, can also cluster and create visible membrane domains. Membrane proteins can be held in place by...

mechanisms of membrane-bending

How Proteins Shape Cell Membranes

Cell membranes can bend into many shapes because they are flexible and made of a fluid mosaic. That bending can be temporary, as in vesicles, or long-lasting, as in microvilli. Cells control the size, place, and time of membrane curvature. Membrane bending can start inside the membrane itself or from proteins attaching to it. Changes in lipid makeup can create curvature, and proteins can help by making the membrane asymmetric or by clustering certain lipids. The shape of integral proteins also...

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

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

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Eukaryotic Compartmentalization

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Prokaryotic Cells

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Cytoplasm

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The Nucleus

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Endoplasmic Reticulum

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Lysosomes

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Peroxisomes

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Introduction to the Cytoskeleton

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Introduction to Actin

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overview-of-myosin-structure-and-function

Overview of Myosin Structure and Function

Myosins are a family of molecular motor proteins, first identified in the skeletal muscles, where they are responsible for muscle contraction. Along with ...

Microtubules

Microtubules are the thickest cytoskeletal filaments with a diameter of 25 nm. In prokaryotic organisms, microtubules are commonly found in locomotory ...

The Structure of Intermediate Filaments

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Types of Intermediate Filaments

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Gap Junctions

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The Extracellular Matrix

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Plant Cell Wall

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pH Regulation in Cells

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Chemistry of the Cell

Introduction to Cellular Respiration

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ATP Energy Storage and Release

ATP and Macromolecule Synthesis

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1. Engage in evidence-based argument to relate a cell's function to the structure, function, and diversity of its components. Examples: Muscle cells have a large number of mitochondria, which create energy for contraction in the form of ATP. Some plant cells have a large vacuole which is used for storage and transport of excess nutrients in changing environments.

9-12.B.1.1

What is Gene Expression?

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RNA Structure

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Types of RNA

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Transcription

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Bacterial RNA Polymerase

Eukaryotic RNA Polymerases

General Transcription Factors

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RNA Polymerase II Accessory Proteins

Transcription Elongation Factors

pre-mrna-processing-modification-of-pre-mrna-ends

Pre-mRNA Processing: Modification of pre-mRNA Ends

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or ...

Pre-mRNA Processing: RNA Splicing

Chromatin Structure and RNA Splicing

Alternative RNA Splicing

Nuclear Export of mRNA

Transfer RNA Synthesis

Ribosomal RNA Synthesis

The Nucleolus

Additional Subnuclear Structures

Translation

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Initiation of Translation

Termination of Translation

Improving Translational Accuracy

prokaryotic-transcriptional-activators-and-repressors

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Nonsense-mediated mRNA Decay

The Eukaryotic Promoter Region

Molecular Chaperones and Protein Folding

Co-activators and Co-repressors

The Proteasome

Master Transcription Regulators

Regulated Protein Degradation

Regulated mRNA Transport

Proteins: From Genes to Degradation

mRNA Stability and Gene Expression

Cell Specific Gene Expression

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between ...

regulation-of-expression-occurs-at-multiple-steps

Regulation of Expression Occurs at Multiple Steps

Small interfering RNAs (siRNA)

Cis-regulatory Sequences

lncRNA - Long Non-coding RNAs

cooperative-binding-of-transcription-regulators

Cooperative Binding of Transcription Regulators

Epigenetic Regulation

Epigenetic changes alter the physical structure of the DNA without changing the genetic sequence and often regulate whether genes are turned on or off. ...

2. Obtain and evaluate information to explain the role of DNA and RNA in transcription and translation leading to protein synthesis and cellular function.

9-12.B.1.2

Nucleic Acids and Nucleotides

Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell&#39;s genetic blueprint and have instructions for its ...

Nucleic Acid Structure

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Genomic DNA in Prokaryotes

The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the ...

Genomic DNA in Eukaryotes

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a. Use a model to describe the structure and sequence of DNA, including nucleotide structure, base pairing, and the structure of the helix.

9-12.B.1.2.a

How Detergents Solubilize Membrane Proteins

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