SFP3 - Protein purification Flashcards
What is protein purification?
The process of isolating an individual protein or protien complex from a mixture, which may contain other cell components including DNA, cell membranes, and other proteins”
How many proteins may a human cell have in its proteome?
Up to 100,000
What is size exclusion chromatography?
Separation of proteins based on their size
What is ion exchange chromatography?
Separate proteins based on their overall charge
How can denaturation be prevented during purififcation?
Low temperatures, physiological buffers used
At what measurement do peptide bonds absorb UV light?
220nm
How does size exclusion chromatography work (gel filtration)?
Column filled with ‘beads’ with small pores. Smaller proteins in native state enter pores and passage impeded to produce a collection of fragments. Proteins can then be analysed to find the fraction containing protein of interest.
What does ion-exchange chromatography depend upon?
Relative numbers of charged R-groups and the pH of the buffer
How does it work?
Column of beads are charged, either positive or negative, will bind to oppositely charged proteins
How does affinity chromatography work?
A ligand with a high binding affinity for the protein to be purified is covalently attached to beads. Proteins pass through the column and proteins of interest are ‘affinity-purified’
