In general, separation techniques focus on what?
size, charge, and polarity
As the purification steps are followed, we make a what to track the succes?
make a table of the recovery and purity of the protein
In this sort of table, what does the percent recovery colum track?
tracks how much of the protein of interest has been retained at each step
What happens to the percent recovery during purification?
it drops steadily
What does the specific activity column compare? what should happen to this number if the purification is successful?
the purity of the protein at each step; this value should go up
A detailed study of the properties of any one protein requires what?
a homogenous sample consisting of only one kind of molecule
What has to happen before the real purification steps can begin?
the protein must be released from the cells and subcellular organelles
What is this first step called?
homogenization = involves breaking open the cells
What is the simplest approach to homogenization?
grinding the tissue in a blender with a suitable buffer, the cells are broken open releasing soluble proteins
This approach also breaks what?
many of the subcellular organelles
What is a gentler technique to releasing the proteins from the cell?
use a Potter-Elvehjem homogenizer, a thick-walled test tube through which a tight fitting plunger is pased, the squeezing of the homogenate around the plunger breaks open cells, but it leaves many of the organelles intact
What is another technique of homogenization?
sonication = involves using sound waves to break open the cells
Cells can also be ruptured by cycles of ________ and _______.
freezing and thawing
What may have to be added if a protein is solidly attached to a membrane?
detergents to unattach it
After the cells are homogenized they are subjected to what?
differential centrifugation
Spinning the sample at 600 times the force of gravity (600 x g) results in what?
a pellet of unbroken cells and nuclei
What happens if the protein of interest is not found in the nuclei?
that precipitate is discarded and the supernatant can then be centrifuged at higher speed
What does 15,000 x g collect in the pellet?
the mitochondria
Further centrifugation at 100,000 x g results in what?
bring down the microsomal fraction in the pellet, consisting of ribosomes and membrane fragments
What happens if the protein is soluble?
the supernatant from the 100,000 spin will be collected and will already be partially purified because the nuclei and mitochondria have been removed
After the proteins are solubilized, they are often subjected to what? what is the most common reagent to use at this step and what is this procedure referred to as?
a crude purification based on solubility; ammonium sulfate = salting out
What happens when ammonium sulfate is added to a protein solution?
some of the water is taken away from the protein to make ion-dipole bonds with the salts
With less water available to hydrate the proteins, what happens?
they begin to interact with each other through hydrophobic interactions
At a defined amt of ammonium sulfate, what precipitates?
something that contains contaminating proteins which are centrifuged down and discarded
What happens after this step?
more salt is added and a different set of proteins, which usually contains the protein of interest, precipitates, which is collected by centrifugation
Do these preliminary techniques generally give a pure sample?
no, but they serve the important task of preparing the crude homogenate for the more effective procedures