Lecture 9 Biochemistry Techniques

Question 1

Cell fractionation, protein isolation/purification

Answer 1

Cells must be fractionated into soluble and particulate fractions (nucleus, mitochrondria for prokaryotes, etc)
Separate cell in diff. components
Once separated, the protein must be isolated in the fractions
Once protein population is isolated, the individual proteins will be isolated (beta-galactosidase)

Question 2

Homogenization

Answer 2

Process of breaking open cells
Gentle manner, organelles can be isolated
Strong methods can damage organelles

Question 3

Fractionation

Answer 3

Isolation of organelles
Many fractionation procedures are performed depending on the cell of interest (plants are harder to isolate due to cell wall)

Question 4

Homogenization Techniques

Answer 4

1. Osmotic alteration
   - Hypo-osmotic buffer that causes swelling which assist in cell rupture (plasma membrane ruptures but leaves organelles intact)
   - Conc. of ions outside is lower than the inside which causes water to go inside the cell
2. Physical force to disrupt cell structure
   - Mortars and pestles, blenders, compression and/or expansion, or ultrasonication - high frequency sounds
   - Add detergent to aid in disruption Ex. NP40 for gentle, SDS for strong disruption

Question 5

Differential centrifugation

Answer 5

Useful when fractionating eukaryotic cells into subcellular fractions
Repeated centrifugation w/ increasing speeds to isolate various organelles
Cells must be broken open in appropriate buffer, separate on gradient by density

Question 6

Types of centrifuges

Answer 6

Distinguishing features are speed and capacity
1. Microcentrifuge: 1 to 2 mL plastic centrifuge tubes at speeds up to 12 000 xg (g-force)
2. High/Super speed: speed about 20 000 rpm (max 20000 xg)
3. Ultracentrifuge: speeds up to over 70 000 rpm (over 100000 xg)

Question 7

SDS Sample Buffer Components

Answer 7

Tris
- used as buffer to maintain pH of 6.8
SDS (sodium dodecyl sulphate)
- strong anionic detergent
- lyse cells and denature proteins
Beta-mecraptoethanol
- reducing agent
- break disulphide bonds between multi-subunit proteins
Glycerol
- make sample heavy/dense to sink in wells of gel
Bromophenol blue
- tracking dye to monitor gel electrophoresis and track progress