Fractionation and Homogenization 18/11/19
Purpose of homogenization = for cytology examiniation
- Cell = organelles – specific functions
- Electron micrograph = sturcture of organelles – not function
- Isolation of organelles = info on diseases
Fractionation = done in different (8) methods
- Each organelle = marked at specific point of centrigugation
- Each fraction = further studies – for abnormatlities in organelles
Several organelles in cell – living, membrane bound
- Perform esential function – metabolic, biosynthesis, transportation, storage, reproduction
Cell fractionation:
- 3 steps – extraction, homogenization, fractionation
Extraction:
- 1st step – for isolation of sub-cellular structures (organelles)
- Biological activity/structure needs to be maintained – prevents degredation
- Keep pH constant, salt solution needs to be constant – (ph = 7.3)
- Isotonic sucrose solution - 4°C – prevents enzyme activity
Homogenization:
- End product after homogenization = homogenate – 8 methods
- Means to ‘break open’
- Breaks membrane – cytoplasmic and nuclear
- Aim = achieve highest level of cell breakage by using minimum disruptive forces without
damaging organelles
Factors that indicate choice of method:
- Volume/no. cells to be homogenized – mechanical method
- No. samples to be homogenized – mechanical method – large no. samples = expensive
machine
- How difficult it is to homogenize cells – physical, mechanical, enymatic, chemical – processing
lung = different to processing lung – bacteria = cell wall – needs to ensure rupturing takes
place, but blood cells = easy to rupture
- Impact of method on desired product – chemical method – compromization needed – (mild
method = low yield, high quality, harsh method = vice versa)
- How stable is product that is being isolated – mechanical method – some cells = unstable –
method needs to be suitable – e.g. – DNA = stable but RNA ≠ stable
- How dangerous are cells, products, methods – for homogenization of toxic/pathogenic
samples (e.g. – biopsy) – safety = priority
- Cost of method – use cheap, expensive material appropriately
Purpose of homogenization = for cytology examiniation
- Cell = organelles – specific functions
- Electron micrograph = sturcture of organelles – not function
- Isolation of organelles = info on diseases
Fractionation = done in different (8) methods
- Each organelle = marked at specific point of centrigugation
- Each fraction = further studies – for abnormatlities in organelles
Several organelles in cell – living, membrane bound
- Perform esential function – metabolic, biosynthesis, transportation, storage, reproduction
Cell fractionation:
- 3 steps – extraction, homogenization, fractionation
Extraction:
- 1st step – for isolation of sub-cellular structures (organelles)
- Biological activity/structure needs to be maintained – prevents degredation
- Keep pH constant, salt solution needs to be constant – (ph = 7.3)
- Isotonic sucrose solution - 4°C – prevents enzyme activity
Homogenization:
- End product after homogenization = homogenate – 8 methods
- Means to ‘break open’
- Breaks membrane – cytoplasmic and nuclear
- Aim = achieve highest level of cell breakage by using minimum disruptive forces without
damaging organelles
Factors that indicate choice of method:
- Volume/no. cells to be homogenized – mechanical method
- No. samples to be homogenized – mechanical method – large no. samples = expensive
machine
- How difficult it is to homogenize cells – physical, mechanical, enymatic, chemical – processing
lung = different to processing lung – bacteria = cell wall – needs to ensure rupturing takes
place, but blood cells = easy to rupture
- Impact of method on desired product – chemical method – compromization needed – (mild
method = low yield, high quality, harsh method = vice versa)
- How stable is product that is being isolated – mechanical method – some cells = unstable –
method needs to be suitable – e.g. – DNA = stable but RNA ≠ stable
- How dangerous are cells, products, methods – for homogenization of toxic/pathogenic
samples (e.g. – biopsy) – safety = priority
- Cost of method – use cheap, expensive material appropriately
