lab techniques Flashcards

1
Q

what characteristics of proteins can be used to separate them in lab

A
  • size
  • density
  • solubility
  • polarity
  • net charge
  • binding
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2
Q

mobil vs stationary phase in chromotography

A
  • mobile phase and stationary phase have contrasting properties, so molecules will favor sticking to one
  • molecules come out from most affinity to mobile phase to least affinity of mobile phase
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2
Q

how to separate based on density

A
  • centrifuge
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3
Q

salting out

A
  • method of separating by solubility
  • adding salt causes less soluble proteins to come out of solution
  • follow with dialysis using semipermeable membrane to separate bize size
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4
Q

size-exclusion chromatography

A
  • uses porous resin (beads) as solid matrix
  • separates according to size
  • large molecules come out first because they cannot enter the pores and go around
  • surface interactions with the beads may also slow smaller proteins down
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4
Q

ion-exchange chromatography

A
  • uses differences in the sign and magnitude of the net charge of a protein (at a specific pH)
  • cation exchange uses neg charged resin (sticks proteins with pos charge)
  • anion exchange uses pos charged resin (sticks proteins with neg charge)
  • if interaction is strong, need to elute
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5
Q

affinity chromatography

A
  • separates proteins according to affinity for a ligand on the matrix beads
  • antibodies are best if applicable because its highly specific
    ex: use calcium on beads if it’s a calcium binding protein
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6
Q

what does elution mean?

A
  • the process of removing one material from another by washing it with something else
  • can change pH, salt concentrations, or other properties of mobile phase to elute off a column
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7
Q

isoelectric focusing

A
  • use gel strip with immobile pH gradient
  • once the protein sample is applied to the gel, electric current is applied
  • proteins migrate until they reach the pH at which they have no charge (PI)
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8
Q

SDS-PAGE

A
  • form of gel electrophoresis done to separate by weight or size
  • can be used after isoelectric focusing to separate in two dimensions
  • buffer allows electricity to run through while maintaining pH
  • treat with SDS before running gel so that it only separates by size and not charge
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9
Q

enzyme activity vs specific activity

A
  • activity is the total units of enzyme in the preparation
  • specific activity is the total units of enzyme activity per mg of total protein
  • specific activity is a measurement of enzyme purity
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