Protein Isolation Flashcards
Why do we isolate biomolecules?
- physical-chemical properties
- activity and biological roles
- produce synthetic analogues
- develop drugs
- develop applications
general isolation of proteins approach?
- disrupt tissue
- release target protein
- crude fractionation
- fine purification
- confirm and characterize attributes
how are tissues disrupted?
mechanically or enzymatically
how does crude fractionation occur?
precipitation and centrifugation
what is the hardest cell type to lyse?
bacterial cells - very rigid cell wall
extraction buffer examples?
anti-oxid, inhibitors, chelator, bacteriostatic
what do separation and isolation methods rely on?
mass, charge, size, shape. solubility, composition, interactions
sedimentation and fractionation techniques?
chromatography
electrophoresis
mass spec
how is the precipitation of proteins induced?
salts
organic solvents
chaotropic agents
change of pH
why is ammonium sulphate used to ppt proteins?
highly soluble
cheap
pure
doesnt denature
what is a chaotropic agent?
a substance that disrupts the structure and denatures, increases entropy
chaotropic examples?
ethanol, urea, phenol, mg chloride
how do organic solvents cause pptn?
disrupt hydrophobic interactions
how do you measure protein activity?
enzyme kinetics
