lab techniques

Question 1

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

Answer 1

• size
• density
• solubility
• polarity
• net charge
• binding

Question 2

mobil vs stationary phase in chromotography

Answer 2

• 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

Question 2

how to separate based on density

Answer 2

• centrifuge

Question 3

salting out

Answer 3

• 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

Question 4

size-exclusion chromatography

Answer 4

• 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

Question 4

ion-exchange chromatography

Answer 4

• 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

Question 5

affinity chromatography

Answer 5

• 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

Question 6

what does elution mean?

Answer 6

• 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

Question 7

isoelectric focusing

Answer 7

• 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)

Question 8

SDS-PAGE

Answer 8

• 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

Question 9

enzyme activity vs specific activity

Answer 9

• 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