Section 2 Flashcards
What are the physical and chemical properties of proteins?
used for purification and analysis
Mass or size (and shape)
Density
Electrical charge
Binding affinity
What are the 3 main separation methods?
- Centrifugation
- Electrophoresis
- Chromatography
How does centrifugation work?
Particles move depending on their density relative to the density of the fluid
Denser particle than the fluid go to the bottom and for a PELLET, less dense come up to the surface, same density don’t move (particles start dispersed)
What unit is used to measure centrifugal force?
earth gravity in g (abt 10 g)
What are 2 types of of centrfugation set ups?
Swinging bucket rotor: bucket start vertical and swings to horizontal when spins
Fixed-angle rotor: bucket fixed at 45˚ angle
What determines the rate at which the supernatant is cleared of particles at given centrifugal force?
For particle of similar shape, it is determined by the size/mass
“Size” unit calculated this way called Svedberg or S
What is Differential centrifugation?
It is the fact of using different centrifugation speeds to recover different particles
What fluid is usually used in a centrifugation tube?
Sucrose density gradient
Particles of interest stop at a certain specific gradient (layer) in the tube corresponding to their density
What characteristic of proteins is electrophoresis based on?
Charge : mass ratio
Speed of migration determined by net charge/mass ratio
Direction of migration depends on net charge (- go towards + end and inversely)
What is SDS used for?
SDS = anionic detergent sodium docecyl sulfate
Its a hydrophobic tail (12 C) with suflate at one end, one of the O- coupled with Na+
SDS denatures proteins by interaction of hydrophobic tail with hydrophobic amino acid side chains (also binds to itself so coats the polypeptide chain)
Bc all negative, various parts of SDS polypeptide chain repel each other which unfolds the protein
SDS also separated chains of multimeric protein → individual denatured polypeptides
Used in gel electrophoresis
When SDS denatures proteins, there is no difference in shape which could affect the mvt in gel
What is the isoelectric point of a protein?
Its the pH at which the sum of all charges = 0
Low pH = more protons available = more + charges
High pH = less protons = more - charges
Acidic residues are neutral in low pH and negative in high pH (ex: COOH → COO-)
Basic residues are neutral in high pH and positive in low pH (ex: NH3+ → NH2)
The isoelectric point depends on the amino acid composition of each protein
What is isoelectric focusing?
It is a type of electrophoresis
A pH gradient is established using special buffers (ampholytes) immoblized in acrylamide gel
ex: cathode (+) pH 2 → pH 10 anode (-)
Proteins migrate towards their isoelectric point
acidic proteins migrate towards acidic pH, towards the cathode and inversely
Proteins resolve in narrow stripes at each isoelectric point
Explain 2-dimensional gel electrophoresis
- Isoelectric focusing:
All proteins resolve into stripes (separation by charge)
Turn stripes horizontally at the top of an SDS electrophoresis - SDS PAGE (separation by size)
Final result = dots on 2D sheet, helpful bc no relation between pI and molecular weight so reveals simultaneously multiple different proteins
What is the downside of Mass spectrometry?
you can’t re-use the particles as they are destroyed in the process
Which 3 concepts are the basis of mass spectrometry?
- Produce dispersed ions in a gas phase
- Measure acceleration of the ions in electric or magnetic field
- Acceleration depends on mass/charge ration (m/z)
*Molecular weight is specific to each molecule, if charge = 1, m/z = MW
