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Lecture Six:
Protein Isolation
and Purification 9
[Based on Chapter 3
Berg, Tymoczko &
Stryer]
(Figures in red are for the 7th Edition)
 It is possible to separate proteins by differences in their
___________ electric charge
 DEFINITION
 pI
 pI is the Isoelectric Point of a protein. It is the value of
pH when the net surface charge is ZERO for that protein
 Isoelectric Focusing
 This technique requires a pH gradient gel
 It uses a gel of polyampholytes
 Polyampholytes are small multi-charged
polymers with different values of pI
 Applying an electric field to this gel creates the pH
gradient
 Proteins have _________________ of acidic and basic
residues
 They often have an overall positive or negative charge
 Figure 3-11, page 73 (3-11, page 75)
 Proteins placed into the gel move within the applied
electric field according to their surface charge
 When the _______________ equals the pI value for any
given protein it will cease to move in the field
 It is isoelectrically focused
 A ______________ degree of separation can be obtained
 Figure 3-4, page 70 (3-4, page 72)
 Ion-exchange Chromatography
 Ion-exchange chromatography uses beads with a surface
charge chemically attached to them
 This can be a negative (________________ - CM)
OR positive (Diethylaminoethyl - DEAE) charge
 The beads are typically cellulose or agarose
 Example: Proteins with positive charge will attach to
negatively charged beads
 Other proteins will pass down the column unhindered
 The positively charged proteins can then be eluted from
the column
 Eluting = ____________
 Eluting the bound proteins
 Add a low concentration of a salt
 As an example: sodium chloride
 Sodium ions are clearly very strongly positive
 The sodium ions ____ to the beads ______ of the proteins
 Weakly positive proteins will elute off first
 Increasing the salt concentration will cause more
positively charged proteins to elute from the column
 All the positively charged proteins can be collected as
fractions as they come off the column
 The other way round would work too
 Positive beads can be used to separate negatively
charged proteins
 Figure 3-7, page 71 (3-7, page 74)
 Gel Electrophoresis
 Gel Electrophoresis separates proteins according to their
____ by applying an electric charge through a polymer gel
 A polyacrylamide gel is almost always used
 The technique is known as:
Polyacrylamide Gel Electrophoresis ==> PAGE
 Polyacrylamide is chemically inert
Figure 3-7b, page 71 (3-7b, page 74)
 The gel forms as ‘SPAGHETTI-LIKE’ STRANDS
 The most common form of gel electrophoresis uses:
Sodium Dodecyl Sulphate ==> SDS
 Hence SDS-PAGE
 SDS is an _________________
 It disrupts all non-covalent interactions in proteins
 SDS binds to amino acid residues in a ratio
of around 1:2
 All proteins become negatively charged
 The negative charge on ____________ becomes directly
proportional to its mass
 -Mercaptoethanol is added to disrupt disulphide bonds
if there are any present
 The proteins are now _____________________
 Figure 3-7a, page 71 (3-7a, page 74)
 The protein mixture flows down the gel from the
cathode towards the anode
 Large proteins are impeded in the gel by the strands
 The smaller proteins pass easily between the strands
 Summarise:
Smallest proteins fastest through the Strands
 Figure 3-9, page 73 (3-9, page 75)
 The protein separation provides a direct measurement of
their ________
 Proteins with known molecular masses are run as a
scale marker beside those with unknown mass
 Differences in mass of ~2% between proteins can be
determined using SDS-PAGE
 Around 10 residues difference
 Affinity Chromatography
 Affinity Chromatography makes use of the fact that many
proteins tightly bind small specific molecules as part of
their function
 Figure 3-5, page 70 (3-5, page 72)
 Example: Concanavalin A
 Concanavalin A binds glucose very tightly
 It is possible to covalently attach glucose to beads
in a column
 Pass the crude protein mixture containing Concanavalin A
down the column
 The Concanavalin A will bind to the glucose on the beads
 All remaining proteins will pass through unhindered
 Concanavalin A can then be removed by passing a
concentrated solution of glucose down the column
 Concanavalin A binds better to the ‘free’ glucose
than to the ‘bound’ glucose on the beads
 The Concanavalin A will elute from the column
bound to the ‘free’ glucose
 Typically the ‘free’ glucose would be
removed by ________
 General Technique
 A protein, Y, recognises and binds a small molecule, X
 Covalently attach X to beads and place these in a column
 In a mixture of proteins containing Y only that protein will
bind to X on the beads and will be retained by the column
 The rest of the proteins will pass on through the
column
 Passing down the column a concentrated solution of ‘free’
X will remove protein Y from the column
 Protein Y will be removed as pure protein
 Summary of Lecture Six:
 Proteins can be separated due to differences in their
surface electric charge
 Isoelectric Focusing
 On a pH gradient gel with an applied electric field, a
protein will move within the field until the charge on
a protein is zero
 This is its Isoelectric Point (pI)
 Proteins can be separated this way
 Ion-exchange Chromatography
 Beads on a column with a given attached surface
charge will bind proteins with an opposing charge
 The bound proteins are then separated and removed
by the addition of a gradually increasing
concentration of a salt
 Gel Electrophoresis
 Most common form of the technique is:
 SDS PAGE
 Separates proteins both by size and charge
 This technique can measure a proteins mass
 Affinity Chromatography
 Separates proteins with specific binding for a small
molecule attached to beads on a column
 The protein is eluted off by passing a
concentrated solution of the ‘free’ small
molecule down the column