KH6 Flashcards
What are the physical and chemical properties of protein purification and analysis
Mass or size and shape
Density
Electrical charge
Binding affinity
What are separation methods in protein purification and analysis
Centrifgation
Electrophoresis
Chromatography
How is the centrifugal force that the centrifuge tube generates measured
In units of earth’s gravity (/g)
What does centrifugal force in centrifuges act on
Particles (down to molecular size) suspended within the liquid medium of the centrifuge tube
What kind of particle will be found at the bottom of the tube and why
Particles denser than the suspending medium will be pushed to the bottom by the g force
What kind of particles will be found at the top of the tube and why
If the particles are less dense than the suspending medium, the g force will float them
What happens when the particles are the same density as the suspending medium
They will not move in either direction but stay where they are
What is the rate at which the supernatant is cleared of particles at a given centrifugal force dependant on
The size/mass of the particles
What is the size unit calculated of a particle in a supernatant
Svedburg (S)
What is an examples of differential centrifugation
Separation of cellular contents by particle size/mass (mass of nucleus»_space;> mitochondria)
Low speed centrifugation pellets nuclei, leaves mitos in supernatant
After: higher speed to recover mitos from supernatant
What is the process of purification of coronavirus
Two spins: first to take out mito at low speed, second at high speed to pellet virus particles
Resuspend pellet, apply equilibrium density gradient centrifugation
How does equilibrium density gradient centrifugation work
- Create a density gradient by smoothly mixing high and low density sucrose solutions while filling the centrifuge tube
- Apply the resuspended virus pellet to the top of the sucrose gradient
- Centrifuge at high speed: virus starts to move toward bottom of tube but stops when it hits a solution density equal to its own density
What is the direction of migration in a free solution in electrophoresis
Net charge
What is the speed of migration in a free solution in electrophoresis
Net charge/mass ratio
In what way can migration is gel electrophoresis be impeded by the gel
Larger molecules impeded more than small molecules
What does SDS do
Denatures proteins (unfolding) and coats them uniformly
How does SDS denature proteins
By the interaction of its hydrophobic tail with hydrophobic amino cid side chains disrupting the oil drop structure of proteins
What does the hydrophobic tail of SDS bind to and how does it coat the polypeptide chain in a uniform layer of SDS molecules
Hydrophobic residues and to itself (itself helps coat)
How do the various parts of the SDS polypeptide chain disrupt and unfold the protein
Everything is negatively charged and like charges repel
What are the characteristics of the unfolding and structural disruption caused by SDS
Completely denatures individual polypeptides, separates all the chains of multimeric protein into individual denatured polypeptides
Does SDS has influence on denatured protein shape
No
Why are all proteins negatively charged with about the same charge:mass ration
Because SDS is negatively charged and bunds uniformly to proteins
How does a free solution compare to a polyacrylamide gel compare in electrophoretic mobility of particles and why
FS: all SDS protein complexes would have same electrophoretic mobility
PG: gel matrix impedes larger molecules more
What is the relationship between migration rate in polyacrylamide gel and protein size (polypeptide length)
They are inversely proportional
