cell Bio > Methods to Study proteins > Flashcards

Methods to Study proteins Flashcards

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1
Q

Many methods have been developed to study the structural and functional roles of proteins in cells:

A
  • Centrifugation
  • Column Chromatography
  • Gel Electrophoresis
  • Antibody methods
  • Amino acid sequencing

*x-ray crystallography

* nuclear magnetic Resonance (NMR) spectroscopy

-Amino acid sequencing

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2
Q

soucre of cells

A

* tissue fragments ( expants)

* Cell culture- growing a variety of cell types in vitro

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3
Q

What is a cell/tissue culture?

  • Technique /process of growing eucaryotic or procaryotic cells outside the organism and tissue of origin ( ex vivo)
  • Strict laboratory conditions that mimic native environment conditions
A
  • sterility
  • nutrition-media,sera and supplements
  • temperature
  • humidity
  • sturutural support
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4
Q

culture conditions may vary-have to be adjusted for the cell type

A
  • Allows the study of cells under controlled conditions
  • theoretically cel of any type can be cultured
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5
Q

Types of cultures:

Tissue cultures-

A

growth of tissue explants

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6
Q

types of cultures:

Animal cell culture-

A

-growth of isolated cells most common and widely used primary, established or immortalized

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7
Q

Types of Cultures:

Organ culture:

A

parts of organ or whole organ cultured in vitro

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8
Q

Plant cell culture

A

-principles similar to animal and plant cultures

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9
Q

Bacterial cultures

A

different setup than animal and plant cultures

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10
Q

Viral cultures-

A

require the culture of cells as host

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11
Q

why do we grow cells outside the organisms?

A
  • reasearch
  • uniform cell population
  • Study cell response to a variety of stimuli ( one at a time or multiple factors)
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12
Q

HeLa

A

* the first human cell line growing successfully in citro

* started on 2/8/1951 at the tissue Culture Labroratory at Johns Hopkins University

* cervical cancer, HPV, immortalized

-1954- HeLa cells used by Dr. Jonas Stalk during successful development of Polio Vaccine

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13
Q

Seeding-

A

from currently growing or frozen stock

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14
Q

changing media

A

Every~48 hours depending on cell density and metabolic rate

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15
Q

Subcultivating

A

When confluent, cultures have to be divided

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16
Q

freezing and storing

A

in liquid nitrogen or ultracold freezer ( -90 C)

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17
Q

Cell Fractionation

A

As a first step in preparing cells for study by many diffrent procedures it is neccessary to break the cells open and seperate their major components

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18
Q

Cell Fraction

-Methods to Disrupt Cells:

A

*Homogenization

*Osmotic shock

*Ultrasonication

*Mechanical shear

* Detergent extraction

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19
Q

Centrifugation-

A

seperates cellular components on the basis of size , density or buoyancy using centrifugal forces from a centrifuge

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20
Q

Centrifugation-

Types:

A

* differential centrifugation- Separates on the basis of size

* velocity sedimentation-seperates on the basis of size and shape

* buyant density or equilibrium sedimentation -seperates on the basis of buoyancy

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21
Q

Centrifugation-

Terminology:

A

Pellet-material that collects at the bottom of the centrifuge tube

Supernatant-fluid above the pellet

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22
Q

dIFFERENTIAL cENTRIFUGATION:

A

used to separate cell componet on the basis of their size and density

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23
Q

Differential Centrifugation:

Study These Flashcards
A

The faster the speed and the Longer the time , the smaller the somponents thatwill be pelleted at the bottom of the tube.

24
Q

Differential Centrifugation:

Fractions

Study These Flashcards
A

low speed ( 1,000 x g x 10 min ) - pellets whole cells, nuclei and cytoskeleton

Medium speed ( 20,000 x g x 1 20 min) - pellets mitochondria, lysosomes

High speed ( 80,000 x g x 1 hr)- pellets microsomes , small vesicles

Ultrahighspeed (150,000 x g x 3hr) -ribosomes, large macromolecules

25
Velocity Centrifugation:
Seperates the components of cells into layers on the basis of their density, which is a function of thier size and shape. Cellular componets centrifuged at high speed ( 500,000 x g) through a dense medium (e.g. 5-20% sucrose providees a continuous gradient of density)
26
Velocity centrifugation:
Cellular components seperate into bands on the basis of their density \* Fraction with componets of diffrent density collected at the end of the centrifuge cycle. Punching of the bottem of the centrifuge tube---\> medium dripping into a series of tubes--\> fractions of diffrent density
27
Velocity centrifugation Advantages:
\* seperation of a cell into diffrent fractions in a single centrifuge run \* seperation into a larger number of diffrent fractions thathat differential centrifugation.
28
Sedimetation Coefficient- S
\* characterizes the rate at which a component sediments during velocity centrifugation- function of size and shape \* Provides a useful measure of the relative size of large subcellular compounents and macromolecules \* Determined by measuring the distance a component migrates in a density gradient over time.
29
Sedimentation Coefficient- S Unit of mesurment-
Svedberg unit (S) Examples: Lysosome-9400S Eukaryotic ribosomes -80S Prokaryotic ribosome-70 S tRNA-4S
30
Density Gradient Centrifugation-
Seperates the components of cells into layers on the basis of thier buyant density, independent of thier size or shape
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Density Gradient Centrifugation:
-cellular componets centrifuged at high speed ( \< 500,000 x g) through a dense medium ( e.g. 20-70% sucrose or cersium chloride)
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Density Gradient Centrifugation
Dense medium establises a continuous gradient of density \* cellular component seperate into bands on the basis fo their desity ---\> fractions collected into a series of tubes ( from a hole in the bottem of the centrifuge tube)
33
Column Chromatography
used to seperate macromolecules sepecially proteins
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Column Chromatography \* seperate macrolecules on the basis of how they interact with a solid support as they flow under gravity through a glass or metal column \* molecules that interact with the medium are differentially slowed down \* fractions collected as solvent flows out of the bottem of the column
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Column Chromatography Types of chromatography-
\*Gel filtration- seperates on the basis of size \* ions-exchange seperate on the basis of electrical change \* Affinity -separates on the basis of specific binding -affinity
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Electrophoresis-
\* separates macromolecules ( proteins, nucleic acids) on the basis of theeir ability to move through a gel either in a slab or in tubes driven by an electrical current \* direction of movement determined by the net quality of charges on the molecule and ease of moving through the supporting medium
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Electrophoresis types \*SDS- polyacrylamide gel electrophoresis ( SDS-PAGE) -
-separation of the basis of relative size
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Electrophoresis- types: Isoletic focusing-
seperates on the basis of isoelectric point
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Electrophorisis types 2 dimensiona gel electrophorisis-
seperates on the basis of both size and isoletric point
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SDS- polycrylamide gel electrophoresis ( SDS PAGE)
\* Seperates polypeptides on the basis of size ( molecular weight) \*Primary method used to determine the molecular weight of unknown polypeptides.
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SDS- polyacrylamide gel electrophoresis - Procedure: 1. Proteins are dissolved in a negatively changed detergent sodium dodecyl sulfate ( SDS)
- seperate proteins into poypetide subunits - disrupt secondary structre and gives polypeptides an ellipoidal shape - swamps out the native charge of the polypeptide and gives every polypeptide a large negative charge
42
**SDS-polyacrylamide gel electrophoresis** - **(SDS-PAGE)** **procedure:** **2. Negatively charged polypeptides are forced by an electrical current through a porous gel of polyacrylamide toward the positive (+) pole**
\* the smaller the polypeptide the more easily it moves through the gel. the distance moved in a given time is a linear function of molecular weight
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**SDS-polyacrylamide gel electrophoresis** - **(SDS-PAGE)** ## Footnote **procedure** **3. the gel is stained to revel the polypeptides**
\* polypetides are arrayed from the top of the gel to the bottom in order of decreasing molecular weight
44
**SDS-polyacrylamide gel electrophoresis** - **(SDS-PAGE)** ## Footnote **Procedure** **4. the molecular weight of polypeptides from the sample is determined by comparing the thier location on the gel with the location of standard proteins of known molecular weight**
45
Isoelectric focusing-
seperates on the basis of Isoletric point
46
Isoletic point(pI)- PH at which the protiein has no net charge ( # of positive charges = # of negative charges, will not move in an electric field)
-characteristic value for any protein , sensitive enough to detect diffrences between molecules as small as a single phosphate group ( -PO+) \* proteins electrophoresed in a tube of polyacrylamide gel with a pH gradient (IEF) \* each protein moves to a point in the gradient that corresponds to its isoelectric point and stays there.
47
2- dimensinal gel electrophorisis \* seperates on the basis of both isoelectric point pI ( first dimension ) and size molecular weight ( second dimension)
\* combination od SDS- PAGE and isoelectric focusing \* can resolve over a 1000 proteins on a single gel
48
2- dimensioanl gel electrophorisis steps:
1 Isoelectric focusing - seperation on the basis of charge- done in a tube gel 2. Gel soaked in SDS 3. SDS-PAGE \* gel from insoelectric focusing ( with seperated proteins ) [laced horizontally on the top of an SDS-PAGE slab gel and electrophorisis in direction perpendicular to that in IF 4. Gel stanined to revel location of polypeptides \* can be combined with antibody labeling techniques to identify specific polypeptides= western blotting
49
Proteins- Antibody Methods
\* uses antibodies to lable or purify cellular compounds -Because of thier high specificity of binding to antigens, antibodies have become a proniple tool for studying cells
50
Structure of antibodies: \* proteins with paired binding sites that recognize and bind with high affinity to specifie molecular sequences ( epitopes) on other molecules ( antigens)
\* the antigen- binding sites are located at he ends of the two arms \* Diffrent lables or markers can be covalently attached to the base of the Y-shaped molecule to make them cisible by microscopy or on gels \* High affinity to specific molecular sequesnces ( epitotpes ) on other molecules ( antigens ) basis of body defense against pathogensq
51
Types of Antibodies: Polyclonal-
\* made by injections of purified antigen into an animal \* Antibodies are obtained by drawings and purfying blood \* Antibodies obtained from blood are always a mixture of antibodies directed against many diffrent antigens. \* Large quanities of antibody can be obtained by injecting B cells into the peritoneal cavity of a mouse and drawing out the ascites fluid which will rich in a single antibody.
52
Types of antibodies; Monoclonal \* made by fusing B cells ( from an animal injected with purified antigen ) with a tumor cell in cell culture---\> immortal hybrid cell secreting monoclonal antibody \* antibodies obtained from a single clone are specific for a single epitope ( binding site) on a single antigen
\* large quanities of antibody can be obtained by injectiog B cells into the peritoneal cavity of a mouse and drawing out the ascites fluid which will be rich in a single antibody.
53
Monoclonal and olyclonal antibodies used in a variety of methods \* immunoaffinity column chromatography \* immunocytochemistry - light fluresce and electron microscopy
\* identificaion of molecules seperated by electrophorisis
54
Amino Anic sequencing
provides an analysis of the amino acid sequence of polypeptides
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X-ray crystallography
-revels the three dimensional structure of macromolecules
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X- ray crystallography - position and intensity of each spot in the diffraction pattern contains information about the position of the atoms in the protein crystal
\* computer analysis \* atomic model of the protein
57
Nuclear Magnetic Resonance ( NMR) Spectroscopy
reveals the structure of small molecules and parts of large molecules

cell Bio (14 decks)

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