Lecture 13 Flashcards
Enzymes can be quantified by..
comparing the amount needed to complete a set amount of reaction in a set period of time
1 unit (U) of enzyme will catalyze
1umol in 1 min
Activity in a reaction equals…
U/uL or U/mL
specific activity equals…
activity/[protein]
-U/mg or U/ug
bimolecular purification
breaking cells apart and isolating organelles, proteins, DNA, RNA & lipids for individual studies
(Protein Extraction) Cells, tissue, or organisms are broken apart by…
- sonication
- french press
- osmotic shock
- digestion
- detergents
- homogenization
Proteins removed from a cell may become degraded by which non-physiological conditions?
pH changes, heat denaturation, oxidation, proteolysis
how to avoid pH changes
extract/keep in buffered solutions
how to avoid heat denaturation
keep sample cold and heat only when needed
how to avoid oxidation
reducing agents can be added to prevent spontaneous crosslinking
how to avoid proteolysis
lower temp or add protease inhibitors such as EDTA
assays should be..
- sensitive
- specific
- rapid
- quantitative
yield
total activity after purification / total activity before purification
purity
specific activity after / specific activity before
purification by properties
- solubility (salting out)
- ionic charge
- polarity
- size
- binding specificity
ionic charge
– Ion exchange chromatography
– Electrophoresis
– Isoelectric focusing
polarity
reverse phase and hydrophobic chromatography
size
- dialysis
- size-exclusion chromatography
- gel electrophoresis
binding specificity
affinity chromatography
centrifugation
if the protein is soluble it can be partially purified by removing larger/denser contaminants
salting out
- common and reversible
- protein molecules with insufficient hydration will aggregate and lose solubility
dialysis
desalting; dialysis tubing has pores with a specific molecular weight cut-off that allow smaller molecules (salt) to pass
chromatography
Techniques to separate mixtures based on physical properties such as size or charge; mixed sample needs to have variable affinity
stationary phase
a substance that the compounds to be separated pass
by or interact with
