Purification of subcellular fractions by density- gradient equilibrium centrifugation

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