Protien Purification Flashcards
What is protien purification
When you a purifying a protien to study its function and structure
The strategy used to purify the protiens is based on the properties of the protien and other molecules in the solution
What type of protien do you use for protien purification
Why
A soluble protien such as an enzyme
This is because the activity of an nutmeg is east to detect and we can use its activity to track the enzyme
Can add a distinctive property to the protien such as a affinity tag
What occurs in an activity assay
Give an example
You have an enzyme that catalyses a reaction and forms products that can be detected through spectrophotometry
You want to turn lactate into pyruvate so you use lactate dehydrogenase as an enzyme. This forms pyruvate and NADH
The NADH absorb light at 340nm and nad+ (part of the reactants) doesnt
So using spectrophotometery we can see how much pyruvate was made by detections the amount of NADH
What is one unit of Lactate dehydrogenase activity is defined as
The amount of the enzyme needed to catalyze the conversion of lactate to pyruvate
Which then forms 1 micro mol of NADH per min at 37 degrees
What is specific activity
After quantifying all protiens (not just the one you want to purify) as mg of the entire protien you can measure
You then measure the activity for a certain amount of protien (this is specific activity)
The specific activity increases as the protiens with the activity is purified
What is the steps of protiens purification
Cell lysis
Centrifugation
Metal affinity chromatography
Ammonium sulfate precipitation
Size exclusions chromatography
What is centrifugation
After lysis of the cells, you separate the cell debris from the supernatant, This is separion based on mass (heavier goes lower)
You centrifuge for 30min at 7800g then a pellet of cell debris is formed and a supernatent on top
The supernatant which is full of soluble protien is what we’re interested in during purification
What is an example of affinity chromatography without using metal
Column chromatography
Such as tlc plates and mobile/stationary phases
What is an example of metal affinity chromatography
You add six His residues to the protien
The metal beads have Ni2+, these beads bind to the protien due to the lone pairs on the imidazole group of histidine
What type of bind is it called when a lone pair donates electrons to nickel during metal affinity chromatography
A coordinate or dated covalent bond
How to you displaced the protien from the metal beads in metal chromatography
Lower the ph (the imidazole gets protonated and has no lone pair to donate)
Add and excess of imidazole (replaces the his residues in the protiens so the protiens leaves
What is ammonium sulfate precipitation
Changing the solidity of the proteins depending of the amount of salt (ammonium sulfate) in solution
If there’s no salt the protein is insoluble
If add some salt the protein is “salting in” to the solution (becomes slightly soluble)
If you keep increasing the salt concentration past this point, the protein begins to “salt out” and is no longer soluble, it precipitates out of the solution
What is size exclusion chromatography
It’s also called gel filtration chromatography
it is when you have carbohydrate polymer beads and the protien sample in solution
The smaller molecules of the proteins enters the aqueous space inside the beads, and the larger molecules can’t go through the beads.
Over time,
The larger molecules flow out of the solution first because they don’t go through the beads and the smaller molecules flow out last
In gel electrophoresis, what does sodium dodecyl sulfate (SDS) do
It denatures/ unfolds the protiens with a negatively charged molecule
This coats the protiens in a negative charge
This allows the protien to move from the negative to the postive end of the polyacrylamide gel
Can also add 2-mercaptoethanol to break apart and sulfide bonds
How do you calculate the specific activity for cell free extract
Metal chromatography
Size exclusion chromatography
For each it’s the
total activity (units)/ the total protien (mg)
The units are (units/mg)
