SFP3: Protein Purification Flashcards
What is protein purification?
The process of isolating an individual protein or protein complex from a mixture which may contain other cell components including DNA, cell membranes and other proteins
Why do we need protein purification?
Both research and commercial applications
Research: to study protein structure and functions (provide insights into normal cell function or understand diseases caused by mutation)
Commercial: proteins may have biomedical uses as ‘drugs’
What is most common way to purify proteins?
Chromatography methods are most common
What can you separate proteins based upon?
Size, overall charge and hydrophobicity
What is protein denaturation?
Reversal of protein folding
Disruption of forces which stabilise secondary - quarternary structure
Do you want to normally purify proteins in active or unactive state?
Normally want to purify proteins in native or active (folded) state so important to prevent denaturation during protein purification
What UV light length do proteins absorb?
280nm - aromatic rings in Trp and Tyr
Can also absorb at 220nm
What can you use to analyse protein purification?
SDS page
What is size exclusion chromatography?
Column filled with beads with small pores, smaller proteins enter pores and passage impeded
What can you use to analyse proteins fractions?
Functional (enzyme) assay or immunochemical methods such as ELISA or western blot
To find fraction containing protein of interest
What is ion-exchange chromatography?
Separate proteins according to overall charge - depends upon relative number of charged -R groups and pH of buffer
Column filled with charged beads, samples loaded in low/no salt
Increase salt concentration, selective elation of different proteins by ‘counter-ions’, collect fractiosn
What is affinity chromatography?
A molecule with high binding affinity for protein to be purified is covalently attached to beads
Protein mixture is passed through a column of these beads and proteins of interest are affinity purified
Bound protein can be eluted
Name an affinity chromatography example
Purification of novel UBA domain binding-proteins
Or
Purification by His-GFP by IMAC
How does His-GFP by IMAC purification work?
Plasmid engineered to express polyhistidine-tagged GFP in E.coli
Poly His is not naturally found in proteins, binds Ni++ or Co++
What is ‘multi-dimensional’ purification?
Exploiting multiple properties of proteins, typically need to combine multiple methods of purification due to the complicated proteomes
