Lecture Five: Protein Isolation
and Purification
[Based on Chapter 3
Berg, Tymoczko &
Stryer]
(Figures in red are for the 7th Edition)
 Initial General Overview
 Some experiments require a protein to be isolated and
purified from the bulk material
 Background
 Protein Source Selection
 If possible find a source that contains large amounts
of the desired protein
 If possible find a source that allows easy extraction
and purification of the desired protein
 These aims are seldom possible
 Protein Accessibility
 The protein should be in solution
 It is already accessible if the desired protein is in the
extra-cellular medium
 The cell must be broken (a process called ______) if the
protein is located in the intra-cellular medium
 Protein Stabilisation
 The proteins in solution will denature unless they are
stabilised against:
 Changes in pH ==> Add a buffer to the mixture
 Changes in Temperature ==> Cool the mixture to
~ 4oC
 Protease degradation ==> Add protease inhibitors
 Protein isolation and purification uses differences in the
chemical and physical properties of proteins
 Protein separation use differences in such properties as:
 __________
 ____
 _______
 Specific Binding Affinity
 Usually, more than one step is required to separate
proteins completely one from another
 After each step, a test - (an assay) - must be performed to
see if the desired protein has been purified from others
 PROTEIN ISOLATION - Separation Techniques
 Salting Out
 Salting out uses the solubility differences of proteins
 The solubility of proteins changes with the addition of
ionic salts
 _______________ concentration decreases protein
solubility
 Different proteins will precipitate out at different salt
concentrations
 This results only in ______________________
of the proteins NOT A FULL SEPARATION
 Figure 3-2, page 69 (3-2, page 71)
 Dialysis
 Dialysis uses the size difference between ___________
and ______________________ to separate them
 Dialysis uses a _______________ membrane
 Protein molecules are retained in the dialysis bag
 Small molecules pass through the bag into the
external solution
 Ionic salts and buffering agents can be removed
 Dialysis can to ___________________ for another
 This permits changes to be made in the pH of the
protein solution
 Figure 3-3, page 69 (3-3, page 71)
 Gel-Filtration Chromatography
 Gel-Filtration Chromatography uses the differences in the
______ of proteins
 This technique uses a column of ________ beads
 The beads are formed from of a highly hydrated
polymer gel
 Examples of polymers used:
 Dextran (A polycarbohydrate)
 _______ (Also a polycarbohydrate)
 Polyacrylamide
 The protein mixture is placed onto the top of a column
 The protein mixture will flow down through the
column and will be collected as fractions
 BUT the proteins in the mixture will have a
range of sizes
 Smaller proteins can permeate the pores in the beads
 The flow of the smaller proteins is slowed by the
beads
 Larger proteins cannot enter the beads
 The larger proteins flow faster around the beads in
the column
 The larger proteins will be first through the column
 Summarise:
Biggest proteins first through Beads
 _______________ of proteins can be separated but the
separation is not well resolved unless there is a
____________ between the sizes of the proteins
 Figure 3-15, page 77 (3-17, page 79)
 Ultracentrifugation
 Ultracentrifugation ultimately uses differences in the
_______ of proteins
 Ultracentrifugation employs very high rotation speeds
 Example: 75,000 rpm
 Proteins are separated according to their ____________
___________
 The sedimentation coefficient is proportional to
protein mass
 A heavier protein will sediment down faster in an
ultracentrifuge
 Buoyancy acts against the centrifugal force
 Related to ____________
 One type of Ultracentrifugation
 Zonal Centrifugation
 Also called Band Centrifugation
 Also called Gradient Centrifugation
 This technique requires a density gradient inside the
centrifuge tube
 The density gradient suppresses _________________
inside the tube
 These would prevent effective protein separation
 Figure 3-15, page 77 (3-17, page 79)
 A density gradient can be produced by the mixing
of low- and high-density solutions
 A typical example: 5% sucrose and 20% sucrose
 The protein mixture is placed on the density gradient in the
centrifuge tube
 Ultracentrifugation separates the proteins into bands
 These proteins can then be collected as fractions of the
solution
 Summary of Lecture Five:
 Protein source selection
 Ideally
 Large quantities available
 Easily isolated and purified
 Rarely possible
 Stabilise proteins against denaturing and degradation
 Separate proteins using the differences in chemical and
physical properties
 A series of steps are often required
 Protein separations require assays to determine the degree
of purification after each step
 Salting out
 Protein solubility changes through the addition of salts
 Dialysis
 Can remove small molecules from proteins
 Gel-filtration chromatography
 Separates proteins according to size
 Largest proteins come off the column first
 Ultracentrifugation
 Separates proteins proportionally to their mass
 Makes use of a density gradient to separate the
proteins