Flashcards in Lecture 13 Deck (30):
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...
-U/mg or U/ug
breaking cells apart and isolating organelles, proteins, DNA, RNA & lipids for individual studies
(Protein Extraction) Cells, tissue, or organisms are broken apart by...
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..
total activity after purification / total activity before purification
specific activity after / specific activity before
purification by properties
-solubility (salting out)
– Ion exchange chromatography
– Isoelectric focusing
reverse phase and hydrophobic chromatography
if the protein is soluble it can be partially purified by removing larger/denser contaminants
-common and reversible
-protein molecules with insufficient hydration will aggregate and lose solubility
desalting; dialysis tubing has pores with a specific molecular weight cut-off that allow smaller molecules (salt) to pass
Techniques to separate mixtures based on physical properties such as size or charge; mixed sample needs to have variable affinity
a substance that the compounds to be separated pass
by or interact with
the carrier for the compounds to be separated
chromatography basic steps
volume of column; pir^2h
volume outside beads
volume inside beads