cell fractionation

yum !

give the 4 main steps of cell fractionation:

• sample preparation

• homogenisation

• filtration

• ultracentrifugation

describe the sample preparation stage:

sample is placed in an ice cold, isotonic, buffered solution

why must the solution used in sample preparation be ice-cold?

reduce enzyme activity that might otherwise digest organelles otherwise

why must the solution used in sample preparation be isotonic?

ensures ψ inside and outside organelles is the same, so they do not burst as a result of osmosis

why must the solution used in sample preparation be buffered?

keeps pH constant so that organelle structures are not damaged and enzymes do not denature

describe and explain the homogenisation stage:

• cells are physically broken open using a blender

• this disrupts the plasma membrane, allowing the organelles to be released into the solution

describe the filtration stage:

• mixture filtered to remove cellular debris and tissue fragments

• filtered through a gauze - allows organelles to pass through while retaining larger debris

describe the ultracentrifugation stage:

• filtrate centrifuged at a low speed → heaviest organelles (nuclei) form pellet at bottom of tube, lighter organelles remain suspended in supernatant

• supernatant transferred to new tube and centrifuged at a higher speed → next heaviest organelles (chloroplasts/mitochondria) form new pellet

• transfer supernatant into new tube and repeat entire process, increasing speed each time until all organelles have been separated into distinct layers

give the order of organelles from heaviest to lightest:

• nuclei

• chloroplasts

• mitochondria

• lysosomes

• ER

• ribosomes

what is the pellet?

sediment at bottom of tube - contains heavier organelles

what is the supernatant?

liquid remaining above pellet - contains lighter organelles