Biochem test one (protein purification)
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What was the very first protein purification process based on?
In order for the protein to be soluble it must_______
inquire a sphere of hydration
How do you exploit the protein solubility for protein purification?
Because the different proteins have different solubilities, so you can separate them by this.
Why do we use salt precipitation?
Because the salt has a stronger attachment to water than protein.
What makes water attracted to salt?
It is soluble and has a high charge density
what are three salts that can be used for this process
- Ammonium sulfate
- sodium chloride
- Potassium chloride
Describe the basic process of Salt precipitation
- Take a protein mixture and add around 10% ammonium sulfate
- Spin it, take the supernatant, add 20% ammonium sulfate
- repeat for 30,40 so on. the protein will be in one of these tubes
what is the least soluble protein?
what comes out at a strength of three?
what is the most soluble protein? Why?
Myoglobin, because it's not a normal component of blood it is generally a muscle protein.
what talks the most ammonium sulfate to bring down?
Salt precipitation uses _________ volumes
To replace water with organic solvent it must________
be miscible with water
alcohol and acetone are________
good organic solvents to replace water
Describe the basic process of replacing the water with organic solvent
- add 10% of organic solvent
- Spin-take supernatant
- you separate the proteins out and then you aciate them to determine where the protein lays.
what is the danger when using organic solvents?
it could denature your protein.
why would the organic solvent denature the protein?
because the inside of the proteins stay together because they are hydrophobic if there is no water around they could start unfolding due to the less hostile environment.
What is the advantage of the organic solvents?
It can extract out lipids.
What property(s) does Polyethyianglycal (PEG) have?
hydrophobic and hydrophilic
Why does a protein interact with water?
Because of the charge
If you remove water from a protein you will remove________
When you reach the net charge of protein being zero, what is this known as?
isoelectric point or (PI)
when is a protein least soluble?
at the isoelectric point (PI)
What s cenate precipitation?
when you adjust the pH in discrete units until it leads to precipitation.
this process will not increase purity by a tremendous amount, but it is a first step and it is useful.
Describe, in general, what chromatography is
It's when you have a matrix of some kind and you apply the protein mixture and the proteins will move down the matrix depending on size/shape
the induction for protein to migrate
the force against movement of the protein is_____
what are some examples of a compelling force?
- a flow of water over the matrix
- putting a charge across the matrix
Examples of Retarding forces
- protein may have a specially affinity for the matrix
Paper, Glass coated with silica gel or a column are examples of what?
different types of chromatography matrix's
the greater the net force the ______the movement
What is the set-up of Thin-layer chromatography?
- thin sheet of glass that is coated w/ hydrophlic compound.
- put compound near the bottom and place it into a tank with a beaker saturated with organic solvents.
- seal tank, flip over the beaker and fill bottom of tank
the solution will began to cliumb the glass in thin-layer chromatography by a process known as________
what are the two layers or phases of thin-layer chromatography?
- Stationary phase- one silica gel
- mobile phase- creeping up the plate
The stationary phase is hydro_____ and the mobile phase is hydro____
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