Lecture 11

Produce a homogenate or cell lysate

Cell walls hard to break, vacuoles fragile and has compounds toxic to enzyme activity.

• 1. Grinding: mortar + pestle
• 2. Extrusion: French press ~ tissues squeezed between rollers
• 3. Slicing: Blender
• 4. Sonication
• 5. Enzymatic disruption: Cell wall hydrolases

Plant cells without cell wall.

Most gentle way to isolate organelles.

Use enzymatic disruption.

• 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.

• 1. Gentle lysis of plasma membrane without damage to organelle.
• 2. Reduce lysis of vacuole and release of its toxic compounds.

• 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.

[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.

1. Put in hypotonic media briefly (too long and organelles lyse).

2. Squeeze protoplast through smaller pore mesh using a syringe.

• 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.

Phenole --> Quinol --> Quinone

React w/ nucleophiles and eventually turns to quinol.

Phenol polymerize to form brown pigments that interact with enzymes.

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).

1. Use phenolic adsorption agents which binds to phenol, forming a complex. Add Polyvinyl polypyrolidone PVPP (insoluble)

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.

RCF = (1.119*10^-5)(rpm^2)r

r is radius

[2(r^2)(pp - pm)]/9n * g

• n = viscosity of medium
• r = radius of particle
• pp = density of particle
• pm = density of medium

• 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.

• 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.

Different velocities through gradient

Start with increasing density. Cellular extract is layered on top. Extract migrates to same density as itself.