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What is the table below and where have you seen it before?
I. Source
II. Method of lysing the cells
III. Protecting the protein from degradation
IV. Means to keep track of your protein – activity assay,
protein purification table
V. Early Stages – gentle fractionation steps to handle
largish volumes.
VI. Middle Stages – chromatographic separation(s)
VII. Final (Polishing) Stages
Structural Integrity
denaturation/renaturation
protein folding/re-folding
subunit dissociation/re-association
Choice of Buffer
Buffer
Salt
Detergents (sometimes)
Glycerol, sugars
Metal chelators
Protease inhibitors (usually)
Reducing agents (often)
Ligands (sometimes)
Centrifuge
B) French Press
C) Sonication
http://extranet.libraries.psu.edu/psul/waring/blendor.html
A French Press, but not
the one we are interested in
http://upload.wikimedia.org/wikipedia/commons/9/91/French_press.jpg
This is the French Press we are interested in
http://en.wikipedia.org/wiki/Sonication
http://www.fishersci.com/wps/portal/PRODUCTDETAIL?aid=2819374
III. Protecting the protein from degradation
-Ion-ion
-Ion-dipole (hydrogen bonds)
-Hydrophobic
• relatively inexpensive
Equilibrium Dialysis
VI. Middle Stages – chromatographic separation(s)
A) Ion-Exchange Chromatography
-anion exchange
-cation exchange
B) Affinity Chromatography
-resin contains ligand or substrate covalently attached
-use of an affinity tag (e.g., “His-tag”)
Affinity Chromatography
The basic idea is to use a resin that contains a
molecular fragment that interacts specifically with
the species of interest (or a relatively small class of
compounds that have similar chemical properties).
C) Ultra-filtration/centrifugal concentrators
Ultrafiltration
Use centrifugation to
achieve solvent flux
through the membrane
Applications:
-Desalting and buffer
exchange
-Concentrating and
purifying proteins,
antibodies and nucleic
acids (alternative to
EtOH precipitation)
Centrifugal Concentrators
or
Define the fractional saturation (Y) as follows:
But, since
Y
The binding constant of a protein for a small ligand
may be determined by equilibrium dialysis