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

דטרגנטים מכילים קבוצות ראש הידרופיליות וזנבות הידרופוביים, המאפשרים להם ליצור מיצלות כדוריות; לכן, הם נמצאים בשימוש נרחב כדי לטהר חלבונים ממברנה.
ישנם שלושה סוגים של דטרגנטים – יוניים, לא יוניים וזוויטריונים.
לדטרגנטים יוניים כגון סודיום דודציל סולפט יש קבוצות ראש טעונות, בעוד שדטרגנטים לא יוניים כגון Triton X-100 אינם טעונים.
לדטרגנטים Zwitterionic כמו CHAPS יש קבוצות ראש עם מטענים חיוביים ושליליים כאחד.
דטרגנטים לא יוניים וזוויטריונים משמשים בדרך כלל לטיהור חלבונים מכיוון שהם מאפשרים לחלבון לשמור על צורתו הפונקציונלית. לעומת זאת, דטרגנטים יוניים יכולים לנטרל את חלבוני המטרה.
חלבוני ממברנה אינטגרליים מטוהרים בשלושה שלבים נפרדים. ראשית, מיצלה דטרגנט אינטראקציה עם bilayer החיצוני של הממברנה.
לאחר מכן, הממברנה הוא מסיס, עם היווצרות של שומנים דטרגנט חלבון קומפלקסים.
לאחר הסרת הדטרגנטים, ניתן לטהר את החלבון באמצעות טכניקות כגון כרומטוגרפיית זיקה.

טיהור דטרגנט של חלבוני ממברנה

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דטרגנט משמש לטיהור החלבונים האינטגרליים של הממברנה. החלק ההידרופובי של דטרגנט יכול להחליף את פוספוליפידים של הממברנה תוך מסיסות חלבוני הממברנה. כאשר מונומרים דטרגנטים מגיעים לריכוז מסוים בתמיסה הנקראת ריכוז המיצלה הקריטי (CMC), הם יוצרים מיצלות. מעל CMC, ריכוז של מונומרים הדטרגנט נשאר בשיווי משקל עם המיצלה. מספר מונומרים הדטרגנטים הקיימים ב-CMC משתנה עבור כל דטרגנט, והוא תלוי בגודל ובמבנה של מונומר הדטרגנט. היווצרות CMC מושפעת גם מטמפרטורה ו-pH. המבנה והריכוז של דטרגנט ומבנה המיצלה שלו קובעים את יכולתו להמיס את דו-שכבת הליפידים.

לטיהור חלבון, ריכוז החלבון והדטרגנט צריכים להיות גבוהים מה-CMC. צריך להיות לפחות מיצלה אחת לכל חלבון הממברנה. גם יחס הדטרגנט-ליפידים ממלא תפקיד חיוני בטיהור חלבון, והוא נקבע בשיטת ניסוי וטעייה עבור כל חלבון היעד. חלק מהחלבונים ניתנים למיצוי עם יחס דטרגנט-ליפידים שנע בין 0.1:1 ל-1:1, אך דו-שכבה הליפידית נשארת ללא הפרעה. בעוד ביחסים הנעים בין 1:1 ל-2:1, מולקולות הדטרגנט ממיסות את הממברנה, וכתוצאה מכך מיצלות מעורבבות עם ליפידים, חלבונים ודטרגנטים. אם היחס מוגדל ל-10:1, כל מולקולות הליפידים בקומפלקס הליפיד-חלבון יוחלפו במולקולת הדטרגנט.

בחירת הדטרגנט תלויה בשאלה האם החלבון המטוהר צריך להיות פעיל פונקציונלית או לא פעיל. דטרגנטים עם ראש קוטבי גדול יותר או שרשרת אלקיל ארוכה יותר נחשבים לקלים יותר מדטרגנטים עם ראש קוטבי קטן יותר או שרשרת אלקיל קצרה יותר. הרכב הממברנה משפיע גם על בחירת הדטרגנט. לדוגמה, Triton X-100 פועל על הממברנה הפנימית של החיידק אך אינו יכול להמיס את הממברנה החיצונית. לעומת זאת, דיגיטונין ידוע על עבודתו הספציפית על ממברנת הפלזמה האוקריוטית.

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דטרגנטים משמשים לטיהור החלבונים האינטגרליים של הממברנה. החלק ההידרופובי של חומר הניקוי יכול להחליף פוספוליפידים קרום בעוד ...

Education

JoVE Core

Cell Biology

ביולוגיה של התא

Unit 1

יחידה 1

Membrane Structure and Components

מבנה הקרום ומרכיביו

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

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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The Nucleus

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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

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The Structure of Intermediate Filaments

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Types of Intermediate Filaments

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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

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Eukaryotic RNA Polymerases

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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 ...

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Chromatin Structure and RNA Splicing

Alternative RNA Splicing

Nuclear Export of mRNA

Transfer RNA Synthesis

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The Nucleolus

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Termination of Translation

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Nonsense-mediated mRNA Decay

The Eukaryotic Promoter Region

Molecular Chaperones and Protein Folding

Co-activators and Co-repressors

The Proteasome

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Regulated Protein Degradation

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Proteins: From Genes to Degradation

mRNA Stability and Gene Expression

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Regulation of Expression Occurs at Multiple Steps

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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

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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 ...

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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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