How to separate organelles from cells?

What is the process to separate organelles from cells?

Cell fractionation

Cell Fractionation?

process used to isolate specific organelles from cells for a closer study

Name of the 3 stages?

• Homogenisation

• Filtration

• Ultracentrifugation

 Step 1 Homogenisation

Cells are broken up using a homogeniser (blender), this breaks the plasma membrane & releases organelles into a solution (homogenate)

Where is homogenisation carried out?

in a cold, isotonic, buffered solution

Why in a cold solution?

Slows enzyme activity

Why in an isotonic solution?

prevents osmotic damage to organelles

Why in a buffered solution?

maintains pH to avoid protein/enzyme denaturation

Step 2 Filteration

Homogenate is filtered through a gauze to remove large debris, Organelles remain in the filtered solution (Filtrate)

Step 3 Ultracentrifugation

• filtrate is placed into a tube

• tube is placed in a centrifuge (machine that seperates materials by spinning)

• filtrate is spun in a centrifuge at increasing speeds

• heaviest organelles form a pellet at the bottom

• rest of the organelles stay suspended in the solution above the pellet ( supernatant)

What happens to the supernatant?

is re-spun at higher speeds to isolate lighter organelles

what is the order of separation (heaviest-lightest)

• nuclei

• choloroplast

• mitochondria

• lysosomes

• endoplasmic reticulum

• ribosomes

Step 4

this process is repeated at increasing speeds until all the different types of organelles present are separated out

Acronym to remember the order of organelle separation

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