purification of subcellular fractions by density- gradient equilibrium centrifugation
TRANSCRIPT
Purification of subcellular fractions by density-gradient 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 bygel 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