Chromatography Flashcards
Give the definition of chromatography
Lab technique used to separate mixtures of substances into their components
Why is chromatography important?
Isolates one protein from thousands to study its properties
What are the two phases in chromatography?
Mobile phases (solvent) Stationary phase (paper)
What are the types of properties of proteins that chromatography utilises?
Charge distribution
Molecular size
Solubility
Binding properties
What is salting in?
When low concs of salt are added the protein solubilities increase
How do salt molecules stabilise protein molecules?
Decreasing the electrostatic energy between the protein molecules, which increase the solubility of proteins
How do salt molecules increase the solubility of proteins?
The salt molecules compete with the protein molecules in binding with water
Why do protein molecules tend to associate with each other instead of salt molecules?
Protein-protein reactions become energetically more favourable than protein-solvent reactions
How does column chromatography work?
The compound mixture moves along with the mobile phase through stationary phase and it separates depending on the affinity/degree of adhesion of each component
What does ion exchange chromatography (IEX) do?
Separates larger amounts of materials than gel filtration
What is the IEX stationary phase?
A polymer (matrix/resin) attached with charged group
What do the acidic groups of resin interact with in IEX?
Positively charged proteins and are called cation exchangers
What do cation exchangers bind to in IEX?
Proteins with positive charges
Give an example of cation exchangers in IEX
CM cellulose canon exchangers
What happens in IEX if groups are basic in nature?
They interact with negatively charged molecules called anion exchangers
What is an example of basic groups in IEX?
Diethylaminoethyl (DEAE) cellulose
How do cation exchangers work?
- Get polymer beads with a negative charge
- add protein/ pass through the beads
- proteins move through the column
In IEX cation exchanges, how do proteins with a more negative net charge move?
Move faster and elite earlier
Give the steps in cation exchangers
-proteins bind to an ion exchanger
-wash column with buffer to elute proteins based on how big an affinity thy have
-
What do cation exchangers bind to in IEX?
Proteins with positive charges
What do anion exchangers bind to in IEX?
Proteins with negative charges
What does a high salt elution buffer do?
Increases mobility of remaining bands so they can be eluted
What are other names for gel filtration chromatography?
Size exclusion chromatography or molecular sieve chromatography
What is gel filtration chromatography used for?
Widely used to purify biological molecules in complex samples like blood
What is separation based on in gel filtration chromatography?
Size and shape
What does the column consist of in gel filtration chromatography?
Porous beads of dextran or agarose: Sephandex and sephanese are commonly used commercial preps
What happens if the proteins can’t fit inside the beads in gel filtration chromatography?
They just pass over and are eluted first
What is the elution volume?
The volume of a solvent required to elute a given solute from the column
When is gel filtration chromatography usually used?
Near the end of the purification process
What is affinity chromatography?
Relatively simple and effective molecular technique which isolated antibodies, antigens, hormones or other proteins by taking advantage of their binding affinity for their respective ligand
What does affinity chromatography require?
- Beads matrix
- solution containing substance to be isolated
- ligand that binds specifically to the target proteins
- a wash to elute non-bound substances
- final wash with elution buffer containing a competitive ligand to elute the target protein
What is affinity material made up of?
Inert support + spacer arms + ligand
What must the first wash in affinity chromatography be?
Sufficient solute concentration, temp and pH to elute all unbound impurities
What should the second wash in affinity chromatography contain?
Competitive ligand
What is the second wash in affinity chromatography used for?
Remove competitive ligand by dialysis after its been eluted
What does high performance liquid chromatography allow for?
The used of smaller profile solution for the column material, thus increasing the surface area
What does a high surface area allow in high performance liquid chromatography?
Much better separation of samples based on hydrophobicity
What is the role of the pump in high performance liquid chromatography?
Force the mobile phase through the column at a specific rule expressed in millilitres per minute
What does the injector do in high performance liquid chromatography?
Introduces the sample into the flow stream of the mobile phase
What is a typical sample volume of a high performance liquid chromatography?
5-20 micro litres
In a chromatogram what does the area underneath the peak signals represent?
How much of the thing there is
What are the advantages of high performance liquid chromatography?
Speed High resolution Sensitivity Reproducibility Accuracy Automation
What are the disadvantages of high performance liquid chromatography?
Cost
Complexity
Coelution
What is coelution
Sometimes there are two molecules each time with the same affinity for the mobile phase, so they elute at the same time?
What are the two steps in 2D electrophoresis?
Isoelectric focussing
SDS-PAGE
What does SDS-PAGE stand for?
Sodium dodecyl sulphate- polyacrylamide gel electrophoresis
How does SDS-PAGE work?
Separates proteins by molecular weight so its based on the size of the proteins
What does a western blot use?
Electrophoresis for protein seperation
Where is the sample placed in elecrophoresis?
Onto the negative end of the polyacrylomide gel
What happens after blotting in electrophoresis?
The membrane is blocked in order to prevent and unwanted membrane-blotter interaction to visualise the protein of interest
