Methods to Study proteins

Question 1

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

Answer 1

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

*x-ray crystallography

* nuclear magnetic Resonance (NMR) spectroscopy

-Amino acid sequencing

Question 2

soucre of cells

Answer 2

* tissue fragments ( expants)

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

Question 3

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

Answer 3

• sterility
• nutrition-media,sera and supplements
• temperature
• humidity
• sturutural support

Question 4

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

Answer 4

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

Question 5

Types of cultures:

Tissue cultures-

Answer 5

growth of tissue explants

Question 6

types of cultures:

Animal cell culture-

Answer 6

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

Question 7

Types of Cultures:

Organ culture:

Answer 7

parts of organ or whole organ cultured in vitro

Question 8

Plant cell culture

Answer 8

-principles similar to animal and plant cultures

Question 9

Bacterial cultures

Answer 9

different setup than animal and plant cultures

Question 10

Viral cultures-

Answer 10

require the culture of cells as host

Question 11

why do we grow cells outside the organisms?

Answer 11

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

Question 12

HeLa

Answer 12

* 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

Question 13

Seeding-

Answer 13

from currently growing or frozen stock

Question 14

changing media

Answer 14

Every~48 hours depending on cell density and metabolic rate

Question 15

Subcultivating

Answer 15

When confluent, cultures have to be divided

Question 16

freezing and storing

Answer 16

in liquid nitrogen or ultracold freezer ( -90 C)

Question 17

Cell Fractionation

Answer 17

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

Question 18

Cell Fraction

-Methods to Disrupt Cells:

Answer 18

*Homogenization

*Osmotic shock

*Ultrasonication

*Mechanical shear

* Detergent extraction

Question 19

Centrifugation-

Answer 19

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

Question 20

Centrifugation-

Types:

Answer 20

* 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

Question 21

Centrifugation-

Terminology:

Answer 21

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

Supernatant-fluid above the pellet

Question 22

dIFFERENTIAL cENTRIFUGATION:

Answer 22

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

Question 23

Differential Centrifugation:

Answer 23

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

Question 24

Differential Centrifugation:

Fractions

Answer 24

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

Question 25

Velocity Centrifugation:

Answer 25

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)

Question 26

Velocity centrifugation:

Answer 26

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

Question 27

Velocity centrifugation

Advantages:

Answer 27

* seperation of a cell into diffrent fractions in a single centrifuge run

* seperation into a larger number of diffrent fractions thathat differential centrifugation.

Question 28

Sedimetation Coefficient- S

Answer 28

* 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.

Question 29

Sedimentation Coefficient- S

Unit of mesurment-

Answer 29

Svedberg unit (S)

Examples:

Lysosome-9400S

Eukaryotic ribosomes -80S

Prokaryotic ribosome-70 S

tRNA-4S

Question 30

Density Gradient Centrifugation-

Answer 30

Seperates the components of cells into layers on the basis of thier buyant density, independent of thier size or shape

Question 31

Density Gradient Centrifugation:

Answer 31

-cellular componets centrifuged at high speed ( < 500,000 x g) through a dense medium ( e.g. 20-70% sucrose or cersium chloride)

Question 32

Density Gradient Centrifugation

Answer 32

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)

Question 33

Column Chromatography

Answer 33

used to seperate macromolecules sepecially proteins

Question 34

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

Question 35

Column Chromatography

Types of chromatography-

Answer 35

*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

Question 36

Electrophoresis-

Answer 36

* 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

Question 37

Electrophoresis

types

*SDS- polyacrylamide gel electrophoresis ( SDS-PAGE) -

Answer 37

-separation of the basis of relative size

Question 38

Electrophoresis-

types:

Isoletic focusing-

Answer 38

seperates on the basis of isoelectric point

Question 39

Electrophorisis

types

2 dimensiona gel electrophorisis-

Answer 39

seperates on the basis of both size and isoletric point

Question 40

SDS- polycrylamide gel electrophoresis ( SDS PAGE)

Answer 40

* Seperates polypeptides on the basis of size ( molecular weight)

*Primary method used to determine the molecular weight of unknown polypeptides.

Question 41

SDS- polyacrylamide gel electrophoresis -

Procedure:

1. Proteins are dissolved in a negatively changed detergent sodium dodecyl sulfate ( SDS)

Answer 41

• 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

Question 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

Answer 42

* 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

Question 43

SDS-polyacrylamide gel electrophoresis - (SDS-PAGE)

procedure

3. the gel is stained to revel the polypeptides

Answer 43

* polypetides are arrayed from the top of the gel to the bottom in order of decreasing molecular weight

Question 44

SDS-polyacrylamide gel electrophoresis - (SDS-PAGE)

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

Question 45

Isoelectric focusing-

Answer 45

seperates on the basis of Isoletric point

Question 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)

Answer 46

-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.

Question 47

2- dimensinal gel electrophorisis

* seperates on the basis of both isoelectric point pI ( first dimension ) and size molecular weight ( second dimension)

Answer 47

* combination od SDS- PAGE and isoelectric focusing

* can resolve over a 1000 proteins on a single gel

Question 48

2- dimensioanl gel electrophorisis

steps:

Answer 48

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

Question 49

Proteins- Antibody Methods

Answer 49

* 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

Question 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)

Answer 50

* 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

Question 51

Types of Antibodies:

Polyclonal-

Answer 51

* 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.

Question 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

Answer 52

* 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.

Question 53

Monoclonal and olyclonal antibodies used in a variety of methods

* immunoaffinity column chromatography

* immunocytochemistry

• light fluresce and electron microscopy

Answer 53

* identificaion of molecules seperated by electrophorisis

Question 54

Amino Anic sequencing

Answer 54

provides an analysis of the amino acid sequence of polypeptides

Question 55

X-ray crystallography

Answer 55

-revels the three dimensional structure of macromolecules

Question 56

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

Answer 56

* computer analysis

* atomic model of the protein

Question 57

Nuclear Magnetic Resonance ( NMR) Spectroscopy

Answer 57

reveals the structure of small molecules and parts of large molecules