12 | Centrifugation and Ultracentrifugation Flashcards
Biological structures exhibit drastic increase in sedimentation under acceleration in a __ __
centrifugal field
If we increase radial distance, the velocity of the biological sample will increase. At the same time its increasing frictional drag will be proportional to _____
velocity
In essentiality what is the rate of sedimentation dependent on?
Applied centrifugal field G (cm s–2)
One revolution, how many radians does it equate to?
2* pi rad
What quantity by measurement is rotor speed defined by?
Revolutions per min
What does the nomograph denote?
the convenient conversion between RCF and
speed of the centrifuge at different radii of the centrifugation spindle to a point along the centrifuge tube
What is the common feature of all centrifuges?
the central motor that spins a rotor containing the samples to be separated
Wrt. centrifuges, particles of biochemical interest are usually suspended in a ?
liquid buffer system contained in specific tubes or separation chambers that are located in specialised rotors
By the application of centrifuges, what factors do we have to think about in order to preserve biological function? (4)
optimum pH value, salt concentration, stabilising cofactors and protective ingredients
such as protease inhibitors
The most obvious differences between centrifuges are?
- Max rotor speed
- presence or absence of a vacuum
- potential for refrigeration or general manipulation of the temperature during a centrifugation run
- maximum volume of samples and capacity for individual centrifugation tubes.
What 3 common centrifuges does undergraduate students encounter during introductory practicals?
And up to which centrifugal force can they reach, individualy by approximation?
- Microfuges (to centrifuge small volumes of samples in
Eppendorf tubes) – 10 000xg - large-capacity preparative centrifuges – < 100 000xg
- ultracentrifuges – < 900 000xg
At 100 000xg, what two compounds are not able to be sufficiently sedimented? And what three are?
smaller microsomal vesicles or ribosomes
nuclei, mitochondria or chloroplast
In order to harvest yeast cells or bacteria from large volumes of culture media, high-speed centrifugation may also be used, but with what flow mode and type of rotor?
continuous flow mode with zonal rotors
When using continuous flow mode, what component is used to sediment biological samples?
Continuous flow of medium instead of centrifuge tubes.
Why does preparative ultracentrifugator operate under vacuum?
In order to minimise excessive rotor temperatures generated by frictional resistance between spinning rotor and air
With analytical ultracentrifuges, what two types of specialised optical systems enables the sedimenting material to be observed throughout the duration of a centrifuge run?
- absorption optical system (based on UV/VIS light absorption) below 230 nm possible using high intensity xenon flash lamps
- Rayleigh interference optical system (based on light refraction)
or a combination of both
What type of distributions of macromolecules in solution can be recorded at any time during ultracentrifugation?
Conc.
What three types of rotor types are there?
Fixed angle
“Swing-out”
vertical
What is the difference between zonal and isopycnic centrifugation?
Rate zonal centrifugation is important in separating particles that differ in size but not in their density, whereas the isopycnic centrifugation is important in separating particles that differ in density but not in their size in gradient centrifugation
Which type of rotor should we use if sedimentation rates differ significantly ?
Fixed-angle rotors
For isopycnic separation until which point is centrifugation continued?
Until samples have reached their isopycnic position in a gradient, i.e. at the point in time when the sedimentation rate is zero because the density of the medium and biological samples are equal
Since samples are not separated down the length of the centrifuge tube when we use vertical rotors, why is it so that isopycnic separation time is significantly shorter as compared to swinging-bucket rotors?
Because the samples are separated across the diameter of the tube
In contrast to fixed-angle rotors, near-vertical rotors have a reduced angle, what does this results in?
much shorter run times
What are near-vertical rotors useful for?
gradient centrifugation of biological elements that do not properly participate in conventional gradients
What steps outlines the path of biological samples during a centrifugation run?
- initial acceleration stage
- the main centrifugal separation phase
- de-acceleration
- final harvesting of separated particles in the rotor at rest
Although run times for fixed-angle rotors are reduced, resolution of separated bands during isopycnic centrifugation is less when compared with ?
swinging-bucket applications
Why is it so that swinging-bucket rotors are the method of choice when maximum resolution of banding zones is required?
Since there’s a greater diversity of gradients exhibiting different steepness to be used. such as in rate zonal studies based on the separation of biological particles as a function of sedimentation coefficient.
What type of centrifuge is used for separating subcellular structures?
preparative ultracentrifugation
What type of centrifuge is used for separating large cellular structures?
conventional high-speed refrigerated centrifugation
What is differential centrifugation based on?
differences in the sedimentation rate of
biological particles of different size and density
Following the initial sedimentation of the largest particles of a homogenate, what does it may contain?
various biological structures or aggregates:
1. nuclear fraction
2. mitochondria
3. chloroplasts
4. large protein precipitates (organelles and membrane
vesicles)
are separated into pellet and supernatant fractions
Why is it so that it’s especially important in the case of rigid biological structures after initial centrifuged crude tissue homogenate, to rehomogenise cellular debris pellets followed by further centrifugation?
To increase the yield of membrane structures and protein aggregates released
With respect to the separation of organelles and membrane vesicles, ____differential centrifugation techniques can be conveniently employed to isolate intact mitochondria and microsomes.
crude
Depending on the particular biological application, a great variety of gradient materials are available. What is used for banding of:
A. DNA, isolation of plasmids, nucleoproteins and
viruses?
B. Whole cells and subcellular particles?
C. Membrane vesicles derived from tissue
homogenates?
A.Caesium chloride
B. Dextran
C. DNase-, RNase and protease-free sucrose medium.
If one wants to separate all membrane species spanning the whole range of particle densities, the maximum density of the gradient must exceed the density of the most dense vesicle species
automatic makers: Both step-gradient and continuous-gradient systems
Manual pouring: Stepwise gradient, continuous gradient.
For the optimum homogenisation of tissue specimens, mincing of tissue has to be performed in the presence of a biological buffer system that exhibits
A. the right pH value
B. salt concentration
C. stabilising cofactors
D. chelating agents.
E. The optimum ratio between the wet weight of tissue and buffer volume
F. temperature (usually 4 °C)
G. presence of a protease inhibitor cocktail is also essential to minimise proteolytic degradation
For the optimum homogenisation of tissue specimens, mincing of tissue has to be performed in the presence of a biological buffer system that exhibits ?
A. the right pH value
B. salt concentration
C. stabilising cofactors
D. chelating agents.
E. The optimum ratio between the wet weight of tissue and buffer volume
F. temperature (usually 4 °C)
G. presence of a protease inhibitor cocktail is also essential to minimise proteolytic degradation
Typical values for centrifugation steps are
10 min for 1000xg to pellet: A
10 min for 10 000xg to pellet: B
20 min at 20 000xg to pellet a fraction enriched in: C
1 h at 100 000xg to separate the: D
A. nuclei and cellular debris
B. the contractile apparatus
C. mitochondria
D. microsomal and cytosolic fractions
_____ salt washes can be carried out to remove myosin contamination of membrane preparations.
Mild
How do we further separate microsomal subfractions (crude surface membrane fraction, triad junctions, longitudinal tubules and terminal cisternae membrane vesicles) derived from different muscle membranes?
Sucrose gradient centrifugation using vertical rotor or swinging-bucket rotor system at a sufficiently high g-force
Due to the inevitable problem during subcellular fractionation procedures with cross-contamination of vesicular membrane populations. What is the technical reason for this?
Lack of adequate control in the formation of various types of membrane species during tissue homogenisation
What type of methodology has to be employed if highly purified membrane preparations are needed for sophisticated cell biological or biochemical studies ?
Affinity separations: lectin agglutination method + immunoblot analysis with a mini electrophoresis unit
As biological macromolecules exhibit random thermal motion, their relative uniform distribution in an aqueous environment is not significantly affected by the __ ___ ___. Thus isolated biomolecules in solution only exhibit distinguishable sedimentation when they undergo immense accelerations as in ultracentrifugation.
Earth’s gravitational field
What two types of analytical ultracentrifuge experiments are there?
sedimentation velocity
sedimentation equilibrium
In sedimentation velocity experiments what is recorded? And what complementary info do we get?
- Change in concentrations, where the rate of movement of a boundary between clear solvent and solution per unit centrifugal field yields sedimentation coefficient (being a function of size, i.e. molecular mass, and shape, based on frictional properties)
- monitoring purity: heterogeniety, aggregation and also for evaluation of conformation (oligomeric states) in solution
Based on sedimentation velocity experiments, wrt. the recorded changes in concentration. For heterogeneous systems what does this method give?
Distribution of sedimentation coefficient
