Molecular Seperations Flashcards
Why do we purify molecules
Study properties
Analyse the distribution in body
Use them eg for medical purpose
What are the 2 techniques used to seperate molecules
Chromatography
Gel electrophoresis
How do you acctually prepare intracellular proteins before using chromatography
Using a centrifuge you isolate proteins from the rest of the cell debris eg membrane
Spin at high speeds and the pellet will be cell debris, the supernatant is the proteins/molecules you want to seperate fully
What is g force in terms of centrifugation / ultracentrifugation
Acceleration due to gravity (speed = centrifugal force)
What are 4 properties chromatography exploits to seperate proteins
Charges
Size/shape
Affinity (binding site)
Hydrophobicity
What is the mobile and stationary phase meaning
Mobile phase is the phase in which molecules move (water usually)
Stationary phase is the phase in which molecules don’t move (eg column or paper)
When molecules are in chromatography they pass the stationary phase and those that bind don’t stay mobile phase. Proteins/molecules that keep moving down are in the mobile phase
What are the 2 forms of chromatography
Thin layer chromatography- using paper
Column chromatography- much longer mobile phase - seperate at a bigger scale.
How do molecules move up in TL chromatography
Molecules soluble in the Eg water are part of solvent and move up with it as a solvent
What are the molecules/proteins called when they come out of the column (don’t bind to stationary phase)
The eluate
The protein is eluting down the column
How does size exclusion chromatography seperate proteins (gel filtration)
Porous bead are added to the column
Small molecules move into the beads but larger molecules pass by
Larger molecules = move faster (elute)
What is chromatography which used charges on proteins to seperate called and how does it work
Ion exchange chromatography
If the stationary phase is +ve , proteins with a negative net charge (side chains) will bind to stationary phase
-ve proteins therefore elute
How do you overcome ion exchange chromatography to get the protein you want
Add salt (naCl-) which means cl will compete to bind to stationary phase ,
The proteins then elute
Explain the reverse phase chromatography process (using hydrophobicity)
Proteins with hydrophobic side chains will bind the the surface due to water (polar). This means proteins that aren’t hydrophobic will elute
How do you overcome hydrophobic proteins sticking to the stationary phase
Use ethanol non polar solvent instead
How does affinity binding chromatography work
Molecules with specific binding sites such as antibodies will bind to the stationary phase if there are beads attached to the complementary binding site
They therefore stay in the column
How do you overcome affinity binding chromatography
Add free binding proteins/molecules to bind to the molecules instead of the beads , this allows them to elute
What does IMac stand for and what is it used for
Immobilised metal affinity chromatography.
It is used for proteins that are harder to seperate
How are recombinant proteins used in IMAC to seperate proteins
The proteins are engineered to have added 6-8 histidine residues(bind with metal)
This allows binding to metal such as zinc++ on the column
How is iMac proteins eluted when they’ve bonded with metals
Add imidazole (structure like histidine) which allows histidine to detach and elute the protein
How is gel electrophoresis different to chromatography
No mobile phase- the liquid is in gel
A gel is used to prevent convection currents (no water circling)
The gel allows movement as bands
What controls the speed of molecules in the gel
Pore size of gels. There are usually different sized to stop small molecules moving too fast
What is induced to allow movement in gel electrophoresis
Electric fields. There will be either or both positive or negative electrodes
What is PAGE
Polyacrylimide gel electrophoresis
What does NATIVE PAGE mean
Proteins seperated by size shape and charge
Why are proteins all usually kept at ph 8
so that all proteins have a -ve net charge (lose protons)
This allows for the movement in one direction
Also any higher would denature the proteins
Explain what SDS page is and how it works
SDS molecule is added which adds a negative overall charge of all proteins
It means all proteins are seperated by size not charge
What is added in sds page to break disulfide bonds in proteins
Mercapto ethanol and they’re heated
Does smaller proteins go further in electrophoresis (sds)
Yes, larger proteins appear first
How do you calculate the molecular weight from SDS page
Use log 10 because it’s in a log scale and then x by the scale the protein shows on = MW
What is the gel electrophoresis called when proteins are seperated by charges at certain ph
Iso electric focussing
In IEF what is the point where the protein appears in the gel
The isoelectric point
Where proteins negative and positive charges are equal at a certain ph
No net charge
What is the ph gradient produced by in ief
Ampholytes
2D gel electrophoresis uses both ief and SDS. Explain the process
First the proteins are seperated by ief using the ph gradient
Once Ip is found
They are put in an electric field with a positive electrode at the bottom
The proteins then seperate using size (smallest goes the furthest)
Why would more proteins appear in SDS than ief
Some proteins have the same isoelectric point
Why is adding ethanol to stationary phase called reverse phase hydrophobicity chromatography
Because it is polar so elutes the hydrophobic proteins eventually
