Module 6: protein purification and chromatography

Question 1

protein extraction

Answer 1

cells tissues or organisms are broken apart
- sonication (single cells)
- French press(high pressure to burst cells, organelles are unharmed)
- osmotic shock
- digestion - continously trying to break dwon certain cell walls
- detergents - breaks down cell membranes
- homogenization - force grinding or blender
- the crude preparation must go through a series of steps to remove cellular contaminants

Question 2

stabilizing extracted proteins

Answer 2

• proteins removed from a cell degrade by the non-physiological conditions
• pH changes : extract keep in buffered solutions
• heat denaturation: keeps sample cold, heat only as needed
• oxidation: thiol group on cysteine
• proteolysis : enzymes that break down proteins

Question 3

purification considerations

Answer 3

• to separate the protein/ enzyme you want from everything else you have to be able to track it using assay
• sensitive
• specific
• rapid
• quantitative

Question 4

purification tracking

Answer 4

• activity - how many U of enzyme per mL
• specific activity: how many U per mg protein
• yield: (total activation after purification)/(total activity before purification)
• purity (SA after)/(SA before)

Question 5

purification by physical properties

Answer 5

• solubility - salting out
• charge - ion exchange chromatography, electrophoresis, isoelectric focusing
• polarity - reversed phase (hydrophobic) chromatography)
  -size- dialysis, size exclusion chromotography, gel electrophoresis)
• binding specificity (affinity chromatography)

Question 6

initial purification: centrifugation

Answer 6

• if the protine is soluable it can be paritally purified by removing larger/denser contaminants by differential centrifugation

Question 7

differential precipitation

Answer 7

• the supernatant (liquid at the top of the centrifuge tube) can be further traced to make some of the proteins in the mixtures less soluable (alter the temp, salinity or ph)
• salting out is a common reversible method ( as salt concentration increases the + - ions will compete with the hydrophilic surface amino acids for water
• the protein molecules with insufficient hydration will aggregate and lose solubility

Question 8

dialysis

Answer 8

• de salting
• excess salt must be removed as it reduces solubility and can imoede subsequently isolation steps
• salts can be removed by dialysis tubing has pores with a specific molecular weight cut off that allow smaller molecules (salts to pass)

Question 9

stationary phase

Answer 9

• a substance that the compound to be separated pass by or interact with

Question 10

mobile phase

Answer 10

• the carrier for the compounds to be separated

Question 11

chromotography

Answer 11

• a mixed sample needs to have variable affinity
• some samples may have higher affinity to the mobile phase
• some samples may have higher affinity for the stationary phase
  with different affinities come different rates of migration = separation of the molecules

Question 12

size exclusive chromatography

Answer 12

• porous beads make up the stationary
• several volume concepts are important:
• the volume of the column is Vt
• the volume outside the beads is Vo
• the volume inside the beads is vi
• the volume at which a sample is eluted is ve
• large proteins can not enter the pores of the feeds, so they will migrate around them (travel only in Vo)
• moderate sized proteins enter beads sometimes which increases the volume available and they move more slowly ( travel in Vo and some Vi)
• small proteins can always enter the pores and have access to all the space of the column (there is no Vo just Vt)

Question 13

column chromotography

Answer 13

• pouring: a class tube with a valve at the bottom is filled with a mixture of the stationary and mobile phases
• packing: opening the stopper and letting some liquid run out to make the stationary phase more densly/evenly packed
• loading: the sample of interest is placed on top of the stationary phase
• running/eluting: the sample flows into the column and more mobile phase is added on top to keep the column from drying out
• collecting: samples of defined volumes are caught into tubes at the bottom and saved/tested

Question 14

partition coefficient

Answer 14

• to get reproducible results from variable columns we need a way to standardize elution measurements
• the partition coefficient is the fractions of the pores within the beads available to the sample
  kav = 0= always excluded by beads
  kav = 1 = beads fully accessible
  Kav = (Ve-Vo/Vt-Vo)

Question 15

limitations of size exclusion chromatography

Answer 15

• is Kav is >0.8 or <0.2 you will get poor separation of proteins
• samples become diluted as:
• turbulence from layering sample over gel
• diffusion during travel
• friction differences alon th eglass tube wall
• it is probably optimistic to expect the sample to become diluted 3x

Question 16

ion exchange chromotography

Answer 16

• a column of positively or negatively charged beads forms the stationary phase
  (anion exchange (column is +, targets are -) (cation exchange (column is - targets are +)
• proteins in solution get carried down by gravity and will get slowed by the beads proportionally based on charge
• specific combination of mobile and stationary phases is chosen to retain protein of interest (pH and [salt]
• like-charged or neutral molecules will elute quickly
• the greater the charge on a protein, the slower is will travel down the column
• samples are collected anf each can be tested for the protein of interest
• stepwise (gradient elution)
• alter the ph to reduce the charge of proteins bound to the column
• increase salt concentration to increase competition for binding the stationary phase

Question 17

ion exchange chromatography elution

Answer 17

• gradient elution can be done using a simple 2 beaker, tube and stirbar setup
• as the elution will occur at the top of the column first and work downwards, this is a focusing column

Question 18

affinity chromotography

Answer 18

• specific trap for protein of interest attached to stationar phase
  enzyme - substrate
  receptor - hormone
  anitgen - antibody
  elution can be problematic as the binding is strong
• high purity can be achieved

Question 19

choosing a chromatography method

Answer 19

• for better yeild and purity more specific methods should be choosen
• order of specificity : affinity, ion exchange, size exclusion