BIOL 3113 Lecture Notes - Lecture 6: Nuclear Magnetic Resonance Spectroscopy, X-Ray Crystallography, Column Chromatography
6 Introduction to Methods to Study Proteins
Many methods have been developed to study the structural and functional roles of
proteins in cells:
*Centrifugation
*Column Chromatography
*Gel Electrophoresis
*Antibody methods
*Amino acid sequencing
*X-ray crystallography
*Nuclear Magnetic Resonance (NMR) spectroscopy
Source of Cells
*Tissue fragments (explants)
*Cell culture- growing a variety of cell types in vitro
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:
•sterility
•nutrition - media, sera and supplements
•temperature
•humidity
•structural support
Culture conditions may vary - have to be adjusted for the cell type
-Allows the study of cells under controlled conditions
-What cell types can be cultured?
Theoretically cells of any type
Types of cultures:
Tissue culture
growth of tissue explants
Animal cell culture
growth of isolated cells
most common and widely used
primary, established or immortalized
Organ culture
parts of organ or whole organ cultured in vitro
Plant cell culture
principles similar to animal cell culture
Bacterial cultures
different setup than animal and plant cultures
Viral cultures
require the culture of cells as hosts
Why do we grow cells outside the organisms?
-Research
Uniform cell population
Study cell response to a variety of stimuli (one at a time or multiple factors)
HeLa
*the first human cell line growing successfully in vitro
*started on 2/8/1951 at the Tissue Culture Laboratory at Johns Hopkins University
*cervical cancer, HPV, immortalized
1954 – HeLa cells used by Dr. Jonas Stalk during successful development of
Polio Vaccine
Seeding
From currently growing or frozen stock
Changing media
Every ~48 hours depending on cell density and metabolic rate
Subcultivating
When confluent, cultures have to be divided
Freezing and storing
In liquid nitrogen or ultracold freezer (-90ºC)
Cell Fractionation
As a first step in preparing cells for study by many different procedures it is necessary to
break the cells open and separate their major components
Methods to Disrupt Cells
*Homogenization
*Osmotic shock
*Ultrasonication
*Mechanical shear
*Detergent extraction
Centrifugation
Separates cellular components on the basis of size, density or buoyancy using centrifugal
force from a centrifuge
types:
*differential centrifugation - separates on the basis of size
*velocity sedimentation - separates on the basis of size and shape
*buoyant density or equilibrium sedimentation - separates on the basis of buoyancy
Terminology:
Pellet - material that collects at the bottom of the centrifuge tube
Supernatant - fluid above the pellet
Differential Centrifugation
Used to separate cell component on the basis of their size and density
The faster the speed and the longer the time, the smaller the components that will be
pelleted at the bottom of the tube
Fractions:
Faster the speed, longer the time, smaller it gets
Low speed (1,000 x g x 10 min) - pellets whole cells, nuclei and cytoskeleton
Medium speed (20,000 x g x 20 min) - pellets mitochondria, lysosomes
High speed (80,000 x g x 1 hr) - pellets microsomes, small vesicles
Ultrahigh speed (150,000 x g x 3 hr) - ribosomes, large macromolecules
Velocity Centrifugation
Separates the components of cells into layers on the basis of their density, which is a
function of their size and shape
Cellular components centrifuged at high speed (500,000 x g) through a dense medium
(e.g., 5-20% sucrose, provides a continuous gradient of density)
Cellular components separate into bands on the basis of their density
Fractions with components of different density collected at the end of the centrifuge
cycle. Punching of the bottom of the centrifuge tube medium dripping into a series of
tubes fractions of different density
Advantages:
*separation of a cell into different fractions in a single centrifuge run
*separation into a larger number of different fractions that differential centrifugation
Sedimentation Coefficient - S
*Characterizes the rate at which a component sediments during velocity centrifugation =
function of size and shape
Document Summary
Many methods have been developed to study the structural and functional roles of proteins in cells: *cell culture- growing a variety of cell types in vitro. 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: sterility, nutrition - media, sera and supplements, temperature, humidity, structural support. Culture conditions may vary - have to be adjusted for the cell type. Allows the study of cells under controlled conditions. Study cell response to a variety of stimuli (one at a time or multiple factors) *the first human cell line growing successfully in vitro. *started on 2/8/1951 at the tissue culture laboratory at johns hopkins university. Every ~48 hours depending on cell density and metabolic rate. As a first step in preparing cells for study by many different procedures it is necessary to break the cells open and separate their major components.
