Lecture 19 - Protein purification Flashcards
For enzymes (protein catalysts), the assay usually measures
enzyme activity
Salt concentration is expressed as
ionic strength
At low ionic strength, protein solubility generally
increases with the salt concentration (salting in)
At high ionic strength, protein solubility generally
decreases with the salt concentration (salting out)
Proteins are generally the least soluble at the
pI (isoelectric point)
Uncharged proteins are _______ to the salt concentrations
insensitive
Water-miscible solvents such as acetone and ethanol are good
protein precipitants
The first experimental step in protein purification is to
get it out of the cell and into solution (cell lysis)
Most of the procedures for lysing cells use some variation of___________ followed by ________
crushing or grinding; filtration and/or centrifugation to remove large, insoluble particles
After homogenization, the tissue is fractionated by
several centrifugation steps
After centrifugation, the next step is often
salting out of the proteins
Proteins are generally less soluble at _______ salt concentratinos
higher
Salting out is usually done with
ammonium sulfate
After salting out, salt can be removed by
dialysis
After salting out and dialysis, ________ is usually performed
chromatography
Gel filtration separates molecules based on
size
In gel filtration chromatography, large molecules travel
faster
Ion-exchange chromatography separates molecules based on
charge
In ion-exchange chromatography, bound proteins are eluted by
increasing the concentration of ions that compete with the protein for binding to the column
Hydrophobic interaction chromatography purifies
nonpolar proteins
Affinity chromatography separates molecules based on
their affinity for the column material
In metal chelate affinity chromatography,
a divalent metal ion (2+) is attached to the matrix
In affinity chromatography, the protein can be eluted by
imidazole
