CSB351Y1 Lecture 4: Lecture 4
26 May 2018
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Lecture 4: Isolation and Purification of Viruses
• Centrifugation
- Differential
- Density gradient
➔ Rate zonal
➔ Equilibrium (isopycnic)
Characterization of Viruses
• Biological characterization can often be performed on crude extracts containing cellular debris
• Chemical studies must be performed on purified virus preparations
• Plant viruses accumulate in large amounts in plants
• Animal viruses are different
- Whole animals are not regularly maintained for the purpose of harvesting a virus
- Chicken eggs used to cultivate influenza virus
- Others maintained and harvested from cell culture
• Purification of viruses from hose cell material can pose significant problem
• Viruses often liberated from host cell by grinding or freeze-thawing procedures
1. Viruses are largely formed of proteins and extraction procedure must preserve the integrity of the proteins
2. Viruses possess a defined size, shape, density, etc
- These properties can be used to purify them
3. It is important to maintain the stability of the viruses
- Avoid extreme pHs, high temperature, high salt, denaturing agents, etc
Centrifugation
• F = f x velocity of molecules
• F = force applied to molecules
• f = frictional coefficient (shape – sphere or rod, viscosity of medium)
1. F (force) = Mω2r
- Centrifugal field that the rotating object is subjected to
- M = mass (grams)
- Ω = angula eloity adian/se
= 2/π60XRPM
- r = radial distance (cm) from axis of rotation to particle
2. F = W/gω2r
- M = W/g
- W = weight of particle (grams)
- g = gravitational constant (980 cm/sec2)
• The molecular weight of a particle is a constant
• Its sedimentation coefficient (S) is a constant
- It measures the force applied to particle by centrifuge vs. the viscous
resistance of the medium through which the particle is moving
- Used to characterize its behavior during centrifugation, proportional to the molecular weight (M)
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Document Summary
Lecture 4: isolation and purification of viruses: centrifugation. Characterization of viruses: biological characterization can often be performed on crude extracts containing cellular debris, chemical studies must be performed on purified virus preparations, plant viruses accumulate in large amounts in plants, animal viruses are different. Whole animals are not regularly maintained for the purpose of harvesting a virus. Chicken eggs used to cultivate influenza virus. These properties can be used to purify them. It is important to maintain the stability of the viruses. Avoid extreme phs, high temperature, high salt, denaturing agents, etc. Centrifugation: f = f x velocity of molecules, f = force applied to molecules, f (force) = m 2r f = frictional coefficient (shape sphere or rod, viscosity of medium) Centrifugal field that the rotating object is subjected to. = 2/ 60xrpm r = radial distance (cm) from axis of rotation to particle: f = w/g 2r.
