Cells And Fractionation

Question 1

Function of Golgi body

Answer 1

Forms secretory vesicles and lysosomes

Question 2

ER

Answer 2

Maintains intracellular circulatory system
Act as storage organs
Provide mechanical support

Question 3

Centrosome

Answer 3

Forms the spindle during cell division and helps movement of chromosomes

Question 4

Cell fractionation involves

Answer 4

3 steps: extraction, homogenisation and fractionation

Question 5

First step of fractionation?

Answer 5

Extraction-

Cells or tissues are suspended in a solution of appropriate pH and salt content (usually isotonic sucrose)

Question 6

Second step of fractionation- ?

Answer 6

Homogenisation: means to break open

Cell breakage without damage to organelles

Question 7

What do you have to consider when choosing the homogenisation method?

Answer 7

1. No of cells/ samples to be homogenised
2. How difficult are the cells to be homogenised
3. what impact will the method have on the desired product
4. How stable is the product being isolated
5. How dangerous are the methods/cells being used
6. Cost of the method?

Question 8

What type of homogenisation methods are there?

Answer 8

Mechanical and non-mechanical 
Mechanical: solid sheer (eg: bead mill)
Liquid sheer (ultrasonication, French press) 

Non mechanical: -physical(thermolysis/osmotic shock)
Chemical (antibiotic/detergents)
Enzymatic (lytic enzymes)

Question 9

Bead mill

Answer 9

Mechanical solid shear that agitate beads at high speeds to break up material
Agitators are fitted with the shaft which provide kinetic energy to the small beads
Disruption takes place due to the grinding action of the rolling beads

Choice of bead is dependent on the type of cell

Question 10

Ultra sonication?

Answer 10

Perfect for liquid samples
Mechanical liquid sheer- work by inducing vibration in a titanium probe that’s immersed in the cell suspension
Homogenisation occurs through the ultrasonic forces and cavitation
Rapid formation and collapse of bubbles producing a shockwave and disrupting cell walls by pressure change

Question 11

French press?

Answer 11

Mechanical, liquid sheer- hydraulic pump that drives a piston, piston forces the liquid sample through a tiny valve under high pressure
As he sample passes through the valve, the cells experience shear stress resulting in cellular disruption and decompression (rupture)

Question 12

Thermolysis?

Answer 12

Non mechanical, physical method mainly used for bacteria-
Heating the cells to (50-55c) to disrupt the outer membrane and release periplasmic proteins
Or to 90c- to break the cell wall and release cytoplasmic contents
(Easy and cheap)

Question 13

Osmotic shock?

Answer 13

Non mechanical physical-
Osmotic stress/physiological dysfunction caused by a change in solute concentration around a cell
May result in cells bursting
(At Low conc water entrees the cell and causes it to burst and at high conc water is drawn out)
Only works with animal cells and protozoa (as they don’t have cell walls)

Question 14

Chemical solvent

Answer 14

Non mechanical chemical- used with plant cells
EDTA can be be used to disrupt the cell walls of gram negative bacteria whose cell walls contain lipopolysaccharide
EDTA will leave holes in the cell wall

Question 15

Detergents

Answer 15

Non mechanical chemical- directly causes damage to the cell wall/membrane
Solubilise membrane proteins and lipids, causing the cell to lyse and release it’s contents

Question 16

Enzymes ?

Answer 16

Non mechanical chemical-

Degrade the cell wall components leading to the release of intracellular compounds

Question 17

The 3rd step of cell fractionation is?

Answer 17

Fractionation- carried out using a centrifuge (spinning device)applies high speed forces to seperate materials based on density/size

Question 18

What are the types of centrifugation?

Answer 18

Differential centrifugation: seperation by size

Density gradient centrifugation: seperation by density

Question 19

What are the steps of differential centrifugation ?

Answer 19

The homogenate is initially centrifuges at low speed to form a sediment of the largest organelles at the top
The supernatant is then centrifuged at higher speeds till the desired portion/organelle is obtained

Question 20

What’s density gradient centrifugation ?

Answer 20

Separates particles in which the sample is centrifuged in a medium that gradually increases in density from top to bottom (sucrose used for dentistry gradient)

Question 21

How does density gradient centrifugation work?

Answer 21

The sample is applied in a thin zone at the top of the centrifuge tube on a density gradient (sucrose)
If the density of the particle is the same as the gradient at any point the particle will stop moving otherwise it will move downwards towards the denser region