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Produce a homogenate or cell lysate
Disruption issues unique to plants
Cell walls hard to break, vacuoles fragile and has compounds toxic to enzyme activity.
Techniques of cell rupturing
- 1. Grinding: mortar + pestle
- 2. Extrusion: French press ~ tissues squeezed between rollers
- 3. Slicing: Blender
- 4. Sonication
- 5. Enzymatic disruption: Cell wall hydrolases
Protoplast and function
Plant cells without cell wall.
Most gentle way to isolate organelles.
How to make protoplasts
Use enzymatic disruption.
Disadvantages of protoplast
- 1. Enzymes are not pure
- 2. Enzymes expensive on large scale.
- 3. Low protoplast yields
- 4. Doesnt work for all cell types or for all species.
- 5. Long incubation times.
- 6. Protoplast cannot be stored and are short lived.
Advantages of protoplast
- 1. Gentle lysis of plasma membrane without damage to organelle.
- 2. Reduce lysis of vacuole and release of its toxic compounds.
Procedure for isolation of protoplast
- 1. Add isotonic media containing cell wall degrading enzymes.
- 2. Incubate for 2-24 hours.
- 3. Filtration: Pass cell through mesh of known pore sizes. Only single protoplast pass through.
- 4. Wash protoplast with isotonic buffer to remove enzyme.
Hypotonic ~ function, vacuole effect
[S]in = [S]out, no cell size change.
[S]in < [S]out, cell shrinks, enzyme activity change.
[S]in > [S]out, cell grows and eventually lysis. Vacuole will lyse eventually.
How to isolate organelles from protoplast
1. Put in hypotonic media briefly (too long and organelles lyse).
2. Squeeze protoplast through smaller pore mesh using a syringe.
Composition of homogenization buffer
- 1. Osmoticum: isotonic.
- 2. Buffer: prevent pH change to preserve enzyme activity.
- 3. Protease inhibitors: decoy proteins
- 4. Add compounds to: a) prevent synthesis or b) remove phenolic compounds.
Phenol Oxidase action
Phenole --> Quinol --> Quinone
React w/ nucleophiles and eventually turns to quinol.
Phenol polymerize to form brown pigments that interact with enzymes.
Reducing quinone interaction w/ proteins (different from removing phenolic compounds)
1. Add reducing agents to reduce quinone to quinol. Use VITAMIN C.
2. Inhibit phenol oxidase using EDTA (Cu chelation), DIECA(Cu specific binding), and N2 saturation (lesser O2).
Ways to remove phenolic compounds
1. Use phenolic adsorption agents which binds to phenol, forming a complex. Add Polyvinyl polypyrolidone PVPP (insoluble)
Useful aspects of quinone reactivity w/ amines
1. Leather tanning: quinone binds to collagen to make it more resistant to decay.
2. Hardening/darkening of insect cuticle.
Force = (w^2)r
- w is angular velocity
- R is radius
Components tend to move away from axis of rotation.
Relative centrifugal force
RCF = (1.119*10^-5)(rpm^2)r
r is radius
Velocity of particle in a centrifuge
[2(r^2)(pp - pm)]/9n * g
- n = viscosity of medium
- r = radius of particle
- pp = density of particle
- pm = density of medium
Centrifuge equations say:
- 1. Bigger particles move faster than smaller ones.
- 2. Denser particles will move faster than less dense once.
- 3. Denser solutions will slow particles down.
- 4. Greater viscosity will have particles move slower.
- 5. Higher the g, the faster the particle.
Types of centrifugation
- 1. Differential centrifugation
- 2. Rate zonal centrifugation
- 3. Isopycnic centrifugation
Velocity of particles based on their size and density.
Supernatant: Lower density particles.
Will only separate particles with large differences in size.
Rate Zonal Centrifuge
Different velocities through gradient
Start with increasing density. Cellular extract is layered on top. Extract migrates to same density as itself.