Purification of subcellular fractions by densitygradient equilibrium centrifugation
18.7 Isolation, Purification, and
Fractionation of Proteins (1)
• Protein purification involves the stepwise
removal of contaminants.
– Purification is measured as an increase in specific
activity of a protein.
– Some identifiable feature of the specific protein
must be utilized as an assay to determine the
relative amount of the protein.
Isolation, Purification, and
Fractionation of Proteins (2)
• Selective Precipitation
– At low ionic strength, proteins tend to remain in
solution.
– At high ionic strength, protein solubility decreases.
– Ammonium sulfate is the most commonly used
salt for protein precipitation.
Isolation, Purification, and
Fractionation of Proteins (3)
• Liquid Column Chromatography
– Chromatography includes a variety of techniques
in which a mixture of dissolved components is
fractionated through a porous matrix.
• Components are fractionated between mobile and
immobile phases.
• The greater the molecule’s affinity for the matrix, the
slower its movement.
• High performance liquid chromatography (HPLC) has
greater resolution due to a tightly packed matrix.
Isolation, Purification, and
Fractionation of Proteins (4)
• Ion-exchange chromatography uses ionic
charge as a basis for purification.
– A pH when the number of positive and negative
charges is equal is the isoelectric point.
– Gel filtration separate proteins by molecular
weight.
• A column is packed with cross-linked polysaccharides of
different porosity.
• Proteins small enough to enter the pores are eluted
last.
Ion-affinity chromatography
Gel filtration chromatography
Isolation, Purification, and
Fractionation of Proteins (5)
• Affinity
chromatography
isolates one protein
from a mixture
using a specific
ligand.
– The technique can
achieve near-total
purification in a
single step.
Isolation, Purification, and
Fractionation of Proteins (6)
• Determining Protein-Protein Interactions
– Antibodies establish protein interactions by
coprecipitation.
– The yeast two-hybrid system:
• A DNA binding domain is linked to the gene for one
protein—the “bait” protein.
• An activation domain is linked to genes encoding
possible proteins that interact with the “bait”.
• A reporter gene (lac Z) is only expressed when the bait
and its partner interact.
Use of the yeast
two-hybrid system
Isolation, Purification, and
Fractionation of Proteins (7)
• Polyacrylamide Gel Electrophoresis
– Electrophoresis is based on the migration of
proteins in an electric field.
• In polyacrylamide gel electrophoresis (PAGE), proteins
are driven through a gel matrix.
• Movement of proteins depends on molecular size,
shape, and charge density.
• The progress of the gel can be followed using a charged
tracking dye.
• The positions of the proteins can be visualized through
autoradiography or Western blot.
Polyacrylamide gel
electrophoresis
Isolation, Purification, and
Fractionation of Proteins (8)
• SDS-PAGE
– It is PAGE carried out in the presence of a charged
detergent, sodium dodecyl sulfate (SDS).
– The repulsion between bound SDS molecules
causes the proteins to unfold into a similar shape.
– Proteins become separated solely on the basis of
mass.
Isolation, Purification, and
Fractionation of Proteins (9)
• Two-Dimensional Gel Electrophoresis
– It separates proteins on the basis of both
isoelectric focusing and molecular weight.
• After separation by isoelectric focusing, the gel is
removed and subjected to SDS-PAGE.
• Proteins can then be analyzed mass spectrometry.
• The technique is ideal for detecting changes in the
proteins in a cell under different conditions.
Two-dimensional gel electrophoresis
Isolation, Purification, and
Fractionation of Proteins (10)
• Protein Measurement and Analysis
– The amount of protein can be determined
measuring the amount if light absorbed using a
spectrophotometer.
– Mass spectrometry (MS) measures the mass of
molecules, determines chemical formulas and
molecular structure, and identifies unknown
substances.
Principles of operation of a mass spectrometer
Isolation, Purification, and
Fractionation of Proteins (11)
• During MS:
– Protein fragments are converted to ions and
separated on the basis of mass and charge.
– Fragments are compared to large protein
databases for identification.
18.8 Determining the Structure of
Proteins and Multisubunit Complexes
• X-ray crystallography (or X-ray diffraction)
uses protein crystals.
– Crystals are hit with X-rays, and scattered
radiation is collected on a photographic plate.
– The diffraction pattern provides information about
the structure of a protein.
– The technique is useful in the study of both
proteins and nucleic acids.
X-ray diffraction analysis
Electron density distribution
Combining data from electron microscopy and
X-ray crystallography
18.9 Purification of Nucleic Acids
• DNA purification procedures differ from
protein purification procedures.
– To obtain DNA, nuclei are isolated and lysed.
– DNA is separated from contaminating materials
(RNA and proteins).
18.10 Fractionation of Nucleic Acids
(1)
• Separation of DNA by gel electrophoresis.
– PAGE is used for separation of small DNA and RNA
molecules; large ones are separated by agarose.
– Nucleic acids are separated on the basis of
molecular weight.
Separation of DNA restriction fragments by
gel electrophoresis
Fractionation of Nucleic Acids (2)
• Separation of Nucleic Acids by
Ultracentrifugation
– Velocity Sedimentation is the rate at which a
molecule moves in response to centrifugal force.
• Size of organelles and macromolecules can be
expressed in S (Svedberg) units.
• The S value provides a good measure of relative size.
Techniques of nucleic acid sedimentation
Techniques of nucleic acid sedimentation
Fractionation of Nucleic Acids (3)
• Ultracentrifugation (continued)
– Equilibrium Centrifugation separates nucleic acids
on the basis of their buoyant density.
• This technique is sensitive enough to separate DNA
molecules having different base composition.
18.11 Nucleic Acid Hybridization
• Nucleic acid hybridization is based on the
ability of two complementary DNA strands to
form a double-stranded hybrid.
• The Southern blot technique is based upon
DNA hybridization.
• The Northern blot technique is based upon
RNA-DNA hybridization.
• Hybridization can be used to determine the
degree of similarity between two samples.
Determining the location of specific DNA
fragments using Southern blot
18.12 Chemical Synthesis of DNA
• Chemical synthesis of DNA or RNA supports
many other procedures.
• The chemical reaction linking nucleotides have
been automated.
• A nucleotide is assembled one at a time up to
a total of 100 nucleotides.
• Modifications can be incorporated into the
molecules.
18.13 Recombinant DNA Technology
(1)
• Recombinant DNA molecules contain DNA
sequences derived from more than one
source.
• Restriction endonucleases are enzymes that
function in bacteria to destroy viral DNA,
restricting the growth of viruses.
Recombinant DNA Technology (2)
• Restriction endonucleases:
– Are used to dissect genomes into precisely
defined fragments for further analysis.
– Restriction maps are complete diagrams of the
fragments that result from digestion of a genome
by specific restriction enzymes.
Construction of a restriction map
Construction of a restriction map
Recombinant DNA Technology (3)
• Formation of Recombinant DNAs
– DNA is first cut with restriction enzymes.
– Recombinant DNAs can be formed in various
ways, such as creating “sticky ends” with
restriction enzymes.
– The two components of a recombinant DNA are
linked using DNA ligase.
Formation of a
recombinant DNA
molecule
Recombinant DNA Technology (4)
• DNA cloning is a technique to produce large
quantities of a specific DNA segment.
– The DNA segment to be cloned is first linked to a
vector DNA.
• Bacterial plasmids and bacterial virus are two
commonly used vectors.
Recombinant DNA Technology (5)
• Cloning Eukaryotic DNAs in Bacterial Plasmids
– Plasmids used for DNA cloning are modified forms
of the wild type.
• Cloning plasmids contain a replication origin.
• Cloning plasmids usually carry genes for antibiotic
resistance.
– Recombinant plasmids are introduced into
bacterial cells by transformation.
– Plasmid-containing bacteria are selected by
treatment with antibiotics.
An example of
DNA cloning using
bacterial plasmids
Recombinant DNA Technology (6)
• Cloning using plasmids (continued)
– Cells containing various plasmids are grown into
separate colonies which can be screened for the
presence of a particular DNA sequence.
• Replica plating produces dishes containing
representatives of the same bacterial colonies in the
same position in each dish.
• In situ hybridization uses a labeled DNA probe to locate
the colony having the desired DNA fragment.
Locating a bacterial colony containing a desired DNA
sequence by replica plating or in situ hybridization
Recombinant DNA Technology (7)
• Cloning using plasmids (continued)
– Once the colony has been identified, live cells
from the colony can be grown into large colonies
to amplify the recombinant DNA plasmid.
– The cells can then be harvested, the DNa
extracted and the recombinant plasmid DNA
separated from the larger chromosome by
equilibrium centrifugation.
Separation of plasmid DNA from the main bacterial
chromosome by CsCl equilibrium centrifugation