Centrifugation

How is RCF calculated?

1.118r (n/1000) squared r = radius in mm n = rpm

How much force and time is required to separate the plasma membrane, ER and golgi?

100000g for 60 mins

How much force and time is required to separate the ribosomes?

150000g for 180 mins or 300000g for 150 mins

How much force and time is required to separate mitochondria and chloroplasts?

15000g for 5-20 mins

How much force and time is required to separate nuclei and cells?

600-2000g for about 5 mins

What are rotors made of?

Light corrosion resistant materials such as Al, Ti or ceramic

What are the types of centrifuge?

Low speed benchtop Microfuge High speed Ultracentrifuge

What is density gradient centrifugation?

Where a density gradient is used to filter out sample based on size and density of particles. Sample is very delicate when removed.

Particles suspended in liquid will move depending on what factors?

Applied force Density Particle size and shape Medium viscosity

How can solution density be changed in density gradient centrifugation?

By changing the viscosity

Is it preferred to use differential centrifugation or density gradient centrifugation and why?

Differential centrifugation as DGC has a higher chance of drawing contaminates through the pipette.

What is RCF?

Gravitational force

Can particle, shape & density be changed

No

Fixed angle rotor

Pellets are on the side of the tube Angle is used so more force is used making it more reliable in extracting sample Can only be used in some samples

Swing bucket rotor

Pellets on middle of tube Smaller centrifugal force is used (about 6000g) Good for easy phase separation

What is density gradient centrifugation also known as?

Rate zonal centrifugation

What is centrifugation?

Separation of particles based on size, shape and density

Differential centrifugation

Used to separate organelles from cell homogenates or cells in media Results in supernatant and sedimented pellet. The pellet is not 'pure' so it may require further separation either by velocity sedimentation or zonal sedimentation