01:694:301 Lecture Notes - Lecture 3: Electrophoresis, Affinity Chromatography, Isoelectric Focusing

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Chapter 3 – ‘Exploring Proteins and Proteomes
Purification of Proteins
Proteins can be purified using techniques that involve electrophoresis,
chromatography, ultracentrifugation, and other methods.
o Proteins can be purified according to solubility, size, charge, and bond
affinity
Understand the difference between electrophoresis with and without SDS.
o Electrophoresis – A molecules with a net charge will move in an electric
field;
o Powerful means for separating proteins and macromolecules (DNA, RNA)
o Carried out in porous gels which serves as molecular sieve that enhances
separation
! Molecules smaller than pores move quicker/easily than larger
molecules in gel
o When electric field applied, proteins migrate from negative to positive
electrodes (top to bottom)
! Usually performed in slab of polyacrylamide gel (SDS)
! Under denaturing conditions, proteins can be separated based on mass
First dissolve proteins in SDS, then !-mercaptoethanol to reduce
disulfide bonds
SDS anions bind to AA residues; complex SDS + denatured protein
" large net negative charge
After electrophoresis dye staining is used to help visualize protein
movement
! Graph is inversely proportional to log of mass
o Pure electrophoresis (without SDS) – separation based on ratio mass to charge;
isoelectric point; movement anode or cathode (refer notes)
KNOW what SDS PAGE stands for.
o Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis
What is isoelectric focusing? (Fig. 3.11)
o Separation of proteins based on acidic/basic residues
o pH gradient on gel; protein migrates until it hits pH = pI
! Once protein reaches pI; electrophoretic mobility is zero " stops
! Proteins differing by one net charge can be separated
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Two dimensional electrophoresis – obtain very high resolution separations;
o Proteins spread across top of polyacrylamide gel according to how far they
migrated during isoelectric focus
o Then they undergo electrophoresis in perpendicular direction
! Thus proteins are separated horizontal direction basis of pI and vertical
direction basis of mass.
Dialysis and gel filtration are separations on the basis of size
o Dialysis
! Proteins can be separated from small molecules (i.e. salt)
! Protein placed in dialysis bag in buffer solution
Small molecules diffuse through pores down [] gradient
Protein (greater size than pore diameter) remains in the bag
! Does not distinguish proteins well
Gel filtration can product good separations and can be used to
estimate molecule weight if protein is globular. (molecular exclusion)
o Results in more discriminating separations on basis of size
o Sample is placed at top of column consisting of porous beads
made of insoluble polymer such as dextran or agarose (Sephadex,
Sepharose)
! Small beads can enter but large ones cannot and remain in
solution
These small beads fit into cervices and have longer path;
exit last
Large beads cannot stick into these cervices and have
shorter path
! Note the following glitch with this method " proteins differ
in shape, non-spherical poses problem; overall works well
spherical proteins
Affinity chromatography can be extremely effective way to purify
certain proteins (esp. isolating transcription factors) /(highly
selective)
o Used to isolate protein that recognizes group X by…
! (1) Attaching X or a derivative of it to a column
! (2) Adding mixture of proteins to this column (then wash with
buffer to remove unbound proteins)
! (3) Eluting desired protein by adding high [] of a soluble form
X or altering conditions to decrease binding affinity
High pressure (performance) chromatography – enhanced version of
column technique
Ion (cation) exchange is more useful with amino acids and peptides
than with proteins, and give a rather crude separation (ambiguous
results for proteins)
o Separation based on net charge; cation slowed down by
negatively charged bead, etc.
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