MolBio3 - 26 Flashcards
Name 3 kinds of chromatography
Ion-exchange, gel-filtration, affinity
How does ion-exchange chromatography work?
Matrix filled with ionic charge that retards the movement of molecules of the opposite charge
Draw a diagram of ion-exchange chromatography
How does gel-filtration chromatography work?
Matrix is inert but porous, small molecules are retarded, large are unretarded
Draw a diagram of gel-filtration chromatography
How does affinity chromatography work?
Matrix has specific ligand covalently attached that will bind a specific protein, which is thus retarded
Draw a diagram of affinity chromatography
What is immunoprecipitation?
Affinity chromatography using specific anti-bodies to bind proteins
How does immunoprecipitation work?
Fusion protein attaches to matrix using anti-body. Fusion protein pulls interacting proteins from mixture (pulldown).
What does SDS-PAGE stand for?
Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis
How does SDS-PAGE work?
Individual polypeptide chains form complex with negatively charged molecules of SDS. These migrate through PAG, greater weight = slower migration
How do individual polypeptide chains form complexes with SDS?
Heated with SDS and mercaptoethanol
Describe 2D gel-electrophoresis
Sample is isoelectrically focussed in 1D, then SDS-PAGE in a 2nd dimension, effectively creating a fingerprint gel for the sample
What is isoelectrical focussing?
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
Outline Western blotting
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
How do enzyme-linked antibodies reveal protein locations?
Either produce colour, or light, or both
Outline mass spectroscopy
Peptide digested by trypsin then one of two methods are used to identify the length/content of each fragement based on its mass
Outline the simplest method of mass spectroscopy
Digest peptide fragments’ mass determined, and the profile compared to predicted profiles of genes we know about. Gene can then be identified
Outline the more complex method of mass spectroscopy
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
Outline DNA footprinting
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
How do gel-mobility shift assays work?
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
Outline the yeast two-hybrid screen
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.
What is phage display used for?
Identification of binding sites
What is the yeast two-hybrid screen used for?
Identification of protein-protein interaction
What is the gel-mobility shift assay used for?
Identifying DNA binding domains
Outline phage display
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
