MolBio3 - 26

Question 1

Name 3 kinds of chromatography

Answer 1

Ion-exchange, gel-filtration, affinity

Question 2

How does ion-exchange chromatography work?

Answer 2

Matrix filled with ionic charge that retards the movement of molecules of the opposite charge

Question 3

Draw a diagram of ion-exchange chromatography

Answer 3

Question 4

How does gel-filtration chromatography work?

Answer 4

Matrix is inert but porous, small molecules are retarded, large are unretarded

Question 5

Draw a diagram of gel-filtration chromatography

Answer 5

Question 6

How does affinity chromatography work?

Answer 6

Matrix has specific ligand covalently attached that will bind a specific protein, which is thus retarded

Question 7

Draw a diagram of affinity chromatography

Answer 7

Question 8

What is immunoprecipitation?

Answer 8

Affinity chromatography using specific anti-bodies to bind proteins

Question 9

How does immunoprecipitation work?

Answer 9

Fusion protein attaches to matrix using anti-body. Fusion protein pulls interacting proteins from mixture (pulldown).

Question 10

What does SDS-PAGE stand for?

Answer 10

Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis

Question 11

How does SDS-PAGE work?

Answer 11

Individual polypeptide chains form complex with negatively charged molecules of SDS. These migrate through PAG, greater weight = slower migration

Question 12

How do individual polypeptide chains form complexes with SDS?

Answer 12

Heated with SDS and mercaptoethanol

Question 13

Describe 2D gel-electrophoresis

Answer 13

Sample is isoelectrically focussed in 1D, then SDS-PAGE in a 2nd dimension, effectively creating a fingerprint gel for the sample

Question 14

What is isoelectrical focussing?

Answer 14

Sample placed at either end of gel, across which a stable pH gradient is setup, and current is applied. Sample moves from each end of the gel to the isoelectric point of zero charge

Question 15

Outline Western blotting

Answer 15

2DGE applied to sample, then transferred to a sheet of nitrocellulose where they are washed with specific protein identifying antibodies. It is washed again with an enzyme-linked antibody to reveal the locations of specific proteins

Question 16

How do enzyme-linked antibodies reveal protein locations?

Answer 16

Either produce colour, or light, or both

Question 17

Outline mass spectroscopy

Answer 17

Peptide digested by trypsin then one of two methods are used to identify the length/content of each fragement based on its mass

Question 18

Outline the simplest method of mass spectroscopy

Answer 18

Digest peptide fragments’ mass determined, and the profile compared to predicted profiles of genes we know about. Gene can then be identified

Question 19

Outline the more complex method of mass spectroscopy

Answer 19

Peptide fragments are segmented by mass, then further fragmented by peptide bond. This creates a set of peptides differing in length by one a/a. Comparing the weights of each length can determine the amino acid. Repeat over and over to learn the entire sequence

Question 20

Outline DNA footprinting

Answer 20

DNA tagged at one end with P32, then single strands cleaved randomly. Denaturation and PAGE lays out DNA profile. In presence of DNA-binding protein, there will be a gap, as certain areas are protect from cleavage. This is the DNA footprint

Question 21

How do gel-mobility shift assays work?

Answer 21

Two samples of DNA are made radioactive, and are PAGEd, one just the DNA, one with cell extract. PAGEing makes free DNA rapidly migrate to the other end of the PAG, whilst those fragments bound to proteins are retarded. Number of discrete amounts of DNA on PAG = number of binding sites

Question 22

Outline the yeast two-hybrid screen

Answer 22

Target protein gene added to DNA-binding domain gene, so when expressed, the target protein is attached to the DNA. Candidate protein genes added to DNA-promoting domain genes, with each tagged candidate introduced into the cell individually. If a candidate interacts with the target, the DNA-promoting gene will be brought along, and the regulated gene will be activated.

Question 23

What is phage display used for?

Answer 23

Identification of binding sites

Question 24

What is the yeast two-hybrid screen used for?

Answer 24

Identification of protein-protein interaction

Question 25

What is the gel-mobility shift assay used for?

Answer 25

Identifying DNA binding domains

Question 26

Outline phage display

Answer 26

DNA encoding peptide of interested inserted into phage vector DNA, then transfected into organism. Peptide in the fusion protein displayed on on surface of phage, to interact with environment. Inject library into environment in which you wish to test for binding, allow equilibrium, remove and isolate. Repeat to narrow binding sites down. Purify final binding pool to determine DNA that codes for an environment-binding peptide