Lecture 3

Question 1

Chromatography overview

Answer 1

1. Polar molecules stick to the stationary phase and non polar stay within mobile phase (go further)

Question 2

Types of chromatography

Answer 2

1. Liquid: mobile phase is toluene, methanol or isopropanol and stationary phase is made of silica
2. Gas: mobile phase is small non polar gas, stationary is thin polar liquid: polar sticks which made them not go far
3. Ion: separate proteins based on charge
   3a. Cation exchange: positive proteins stick to negative beads…negative proteins don’t stick and travel far (anion exchange is opposite)
4. Affinity: separates proteins based on protein affinity for specific ligand
5. Size exclusion: separates large molecules based on size or molecular weight
6. Thin layer: less polar molecules have less affinity for polar stationary phase and travel faster
   6a. Rf=distance travelled by compound/distance travelled by solvent

Question 3

Gel electrophoresis overview

Answer 3

1. Used to separate DNA, RNA and proteins based on size and charge

Question 4

Gel electrophoresis types for proteins

Answer 4

1. Denaturing/SDS gel: separate on weight via SDS
2. Reducing SDS PAGE: if needed to get rid of disulphide bonds
3. Native PAGE: distinguishes between multiple diff proteins/subunits (no denaturing or reducing)

Question 5

Electrophoresis: PI focusing

Answer 5

1. Divides based on PI
   1a. High pH denatures giving -ve charge

Question 6

Electrophoresis: Sanger sequencing

Answer 6

1. Used to determine order of nucleotides on DNA

Question 7

PCR

Answer 7

1. An amplification process to make copies of a specific piece of DNA
2. Steps
   2a. Denature at 95C
   2b. Annealing at 50-70C
   2c. Extension at 70C
   2d. Repeat

Question 8

CDNA

Answer 8

1. DNA is synthesized from mRNA via reverse transcriptase

Question 9

Blotting:

Answer 9

1. Southern: DNA
2. Northern: RNA
3. Western: Protein

Question 10

Radioimmunoassay

Answer 10

1. Use antibodies to determine antigen concentrations in a sample

Question 11

Gram staining

Answer 11

1. Positive: detect purple thick peptidoglycan in gram +ve
2. Negative: detect pink thin peptidoglycan in gram -ve

Question 12

Reducing sugars

Answer 12

1. Benedicts reagent: test for reducing sugars
2. Barfoad’s test: test for monosaccharides
3. Fehlings solution: test for monosaccharides and aldehydes