Transcript Extraction and Purification

Extraction and Purification
proteins
Selection of Tissue
• Choice is based on the type of study
• Usually select tissue that has large amounts of
the materials necessary for the study.
• Tissues are acquired in a fashion that provides
for protection of the system to be studied.
– Use fresh tissue or immediately placed in ice cold
solution
– Bacterial cells are centrifuged at 16,000g and
separated from growth media. The pellet(bacterial
cells) is resuspended in buffer.
Tissue and cell disruption
• There are several method to disrupt cells
and tissues depending on your tissue
source, toughness, and what you wish to
obtain
• When the membrane is disrupted, two
things are added
– Buffer to main pH and compound dissolved in
it to support cell osmotically
– Antioxidant – dithiothreitol or βmercatoethanol
Vigorous methods in total
disruption of membranes
• French press: A hydraulic pressure with special sample
cell. Cells are pressured (20000 psi) and released
through small orifice. This results in a shock wave that
causes the cell to burst. Can choose right side out or
inside out opening. This is the gentlest method.
• Sonication: Uses the same principles as french press.
Instead of a single shock wave there is a continuous
flow of shock waves that causes the cells to burst
repetitively. This results in mixed membrane vesicles.
Often has higher yield than French press, and this
system requires cooling samples. Microwaves are used
and the samples are pulsed and cooled several times.
Vigorous methods in total
disruption of membranes
• Polytron, tissumizer: Consists of rotating blade
within a shaft that can be forced into the cell
suspension or tissue suspension. The technique
uses shear forces to disrupt cells
• Bead Mill, shaker: cells are broken in a container
with beads. The container is shaken vigorously.
The system can generate considerable heat and
so usually has cooling coils around it.
Moderate method for cell or tissue
disruption
• Blender: This is classical waring blender.
Used mostly for plant and connective
animal tissues.
• Grinding with abrasive: Cells are placed
with sand or some other abrasive material
and ground in mortar and pestle. Good
method for tough cells like yeast but is
tedious
Gentle disruption
• Osmotic shock: Mainly for cells without cell wall.
(for cell wall digest with enzymes or snail gut
juice before processing)
• Chemical solubilization: Cell membrane
disrupted with detergents, such as triton x100,
sodium cholate, or lysophospholipids or
enzymes such as lysozymes, zymozymes ,
lipase
• Homogenizer: Mechanical disruption. A ground
glass piston fits in ground glass cylinder. Piston
moved up and down to rupture cells. Usually has
water cooling system
Separation of large parts
• Once cells are disrupted, either we have to
collect parts we need or remove unwanted parts.
– FILTRATION
• Cheese cloth: mainly removes materials that are not
homogenized
• Screens: calibrated wire screens can separate broken and
non-broken eukaryotic cells
• Filters: Variety of filters are available from 0.22µm to some
that will allow proteins molecules less than 3000 to pass are
available. Problem – plugging can be reduced by tangential
flow.
Separation of large particles
• Centrifugation: Accelerated sedimentation.
Sedimentation rate depends on several
factors.
– Force pushing down due to gravity F= mg
– Opposing forces
• Buoyancy density of the particle and the buoyant
force or the density of the solvent
• Frictional force depends on shape of the particles
and the material with which it is going.
Types of centrifuges
• Analytical: used to determine the size of the
molecule being studied
• Preparative: More common type. Has different
type of motors
– Fixed angle: most common with fixed angle. The
sample tube is in a fixed angle. The force (x g) are
reported for the average distance.
– Swinging bucket The buckets are able to swing up in
to a position such that the sample goes directly down
the length of the tube. This allows simple gradient.
Samples are at the bottom of the tube rather than on
the side.
Separation of cellular components
• Differential
centrifugation:
Force
Time
sediment
1000g
5min
Eu. cell
4,000g
10min
Chl,cell
debris, nuclei
15,000
20min
Bacteria, mito
30,000
30min
Small
organelle
200,000
1hr
Small vesicle
100,000
3-10hrs
Ribosome
200,000
10-24hrs
Membrane
sheets
Separation of cellular components
• Density gradient centrifugation;
– Exploits the different density of organelles
– Density gradients are formed by using
sucrose as solute
– Can be step gradient or continuous
– Centrifuge for set time at a know force and
determine where your compound is or run it
until it reaches equilibrium. Sample will stop
moving once its density is equal to solvent’s
density.
Precipitation of proteins
Salting out and IEP
Solubility in water
• Proteins usually carry charge due to
hydrophilic amino acids and terminal
amine and acid groups. Because they
have hydrophilic amino acids on their
surfaces that attract water molecules and
interact with them, proteins are soluble in
water solutions.
Solubility in water
• Solubility of proteins is a function of the
ionic strength and pH of the solution.
• Proteins have isoelectric points at which
the charges of their amino acid side
groups balance each other.
Precipitation
• The solubility is also a function of ionic strength
– Salting-in: At low salt concentrations, the presence of
salt stabilizes various charged groups on a protein
molecule, thus attracting protein into the solution and
enhancing the solubility of protein
– Salting-out: As you increase the ionic strength by
adding salt, there is less and less water available for
the protein to dissolve and proteins will precipitate.
Ammonium sulfate
• Ammonium sulfate is the most common salt
used:
– Because it is unusually soluble in cold buffers (our
extractions are kept cold!).
– In research laboratories as a first step in protein
purification because it provides some crude
purification of proteins separating non-proteins and
some unwanted proteins out.
– Because it yields a precipitated protein slurry that is
usually very stable, so the purification can be stopped
for a few hours while the student gets some sleep
Other salts
• Sodium sulfate
• Polyethylene glycol (PEG): organic
polymer that has properties similar to
ammonium sulfate
Change in pH
• If the ionic strength is very low or very
high, protein tend to precipitate at
isoelectric point.
• In our case we add ammonium sulfate in
two steps (reduce co-precipitation) and
then crystallize by changing pH.