3.3 Protein Isolation [HY]

Question 1

Homogenization

Answer 1

• crushing, grinding, or blending
  the tissue of interest into an evenly mixed solution

Question 2

How are proteins and other biomolecules isolated from body tissues or cell cultures?

Answer 2

cell lysis and homogenization

Question 3

Electrophoresis

Answer 3

• works by subjecting compounds to an electric field, which moves them according to their net charge and size
• Negatively charged compounds will migrate toward the positively charged anode, and positively charged compounds will migrate toward the negatively charged cathode.

Question 4

Migration velocity (v)

Answer 4

• Velocity of migration of compounds to anode and cathode in electrophoresis

Question 5

Migration Coefficient

Answer 5

v = Ez /f

• E: electric field strength
• z: inversely proportional to a
  frictional coefficient
• f: depends on the mass and shape of the migrating molecules

Question 6

Polyacrylamide gel

Answer 6

the standard medium for protein electrophoresis

Question 7

How do molecules move in an electric field?

Answer 7

• a molecule will move faster through the medium if it is small, highly charged, or placed in a large electric field.
• molecules will migrate slower (or not at all) when they are
  bigger and more convoluted, electrically neutral, or placed in a small electric field.

Question 8

Polyacrylamide gel electrophoresis (PAGE)

Answer 8

• a method for analyzing
  proteins in their native states
• PAGE is most useful to compare the molecular size or the charge of proteins known to be similar in size

Question 9

Sodium dodecyl sulfate (SDS)

Answer 9

• polyacrylamide gel electrophoresis
• separates proteins on the basis of relative molecular mass alone
• adds SDS, a detergent that disrupts all noncovalent interactions. It binds to
  proteins and creates large chains with net negative charges, thereby neutralizing the protein’s original charge and denaturing the protein. As the proteins move through the gel, the only variables affecting their velocity are E, the electric field strength, and f, the frictional coefficient, which depends on mass. After separation, the gel can be stained so the protein bands can be visualized and the results recorded

Question 10

Isoelectric point (pI)

Answer 10

• The pI is the pH at which the protein or amino acid is electrically neutral, with an
  equal number of positive and negative charges
• For polypeptides,
  the isoelectric point is primarily determined by the relative numbers of acidic and basic amino acids.

Question 11

How are most AA turned to zwitterions?

Answer 11

• For most amino acids, the zwitterion form occurs when the amino group is protonated, the carboxyl group is deprotonated, and any side chain is electrically neutral.
• For arganine and lysine the zwitterion form occurs when the amino group is deprotonated (and therefore electrically neutral), while the nitrogenous side chain is protonated and the carboxyl group is deprotonated

Question 12

Isoelectric focusing

Answer 12

• exploits the acidic and basic properties of amino acids by separating on the basis of isoelectric point (pI)
• The mixture of proteins is placed in a gel with a pH gradient (acidic gel at the positive anode, basic gel at the negative cathode, and neutral in the middle)
• As the protein reaches the portion of gel where the pH is equal to the protein’s pI, the protein takes on a neutral
  charge and will stop moving.
• Moves towards opposite nodes - to + until reaches PI.
• Cathode is negative and basic
• Anode is positive and acidic
• Mnemonic: Anode is A+

Question 13

Chromatography

Answer 13

• uses physical and chemical properties to separate and identify compounds from a complex mixture
• the isolated proteins are immediately available for identification and quantification
• the more similar the compound is to its surroundings (by polarity, charge, and so on), the more it will stick to and move slowly through its surroundings
• preferred over electrophoresis when large amounts of protein are being separated.
• components that have a high affinity for the stationary phase will barely migrate at all; components with a high affinity for the mobile phase will migrate much more quickly
• We can use myriad different media as our stationary phase to exploit different properties such as charge, pore size, and specific affinities

Question 14

Partitioning (Chromatography)

Answer 14

Varying retention times of each compound in the solution results in separation of the components within the stationary phase

Question 15

Retention time (Chromatography)

Answer 15

The amount of time a compound spends in the stationary phase

Question 16

Column chromatography

Answer 16

• a column is filled with silica or alumina beads as an adsorbent, and gravity moves the solvent and compounds down the column
• size and polarity have a role in determining how quickly a compound moves
• the less polar the compound, the faster it can elute through the column (short retention time)
• the solvent polarity, pH, or salinity can easily be
  changed to help elute the protein of interest.
• The more similar the sample is to the solvent (mobile phase), the more quickly it will elute; the more similar it is to the
  alumina or silica (stationary phase), the more slowly it will elute—if at all.
• Each fraction
  contains bands that correspond to different compounds

Question 17

Ion-Exchange Chromatography

Answer 17

• the beads in the column are coated with charged substances, so they attract or bind compounds that have an opposite charge
• After all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column.

Question 18

Size-Exclusion Chromatography

Answer 18

• the beads used in the column contain tiny pores of varying
  sizes. These tiny pores allow small compounds to enter the beads, thus slowing them down.
• Large compounds can’t fit into the pores, so they will move around them and travel through the column faster

Question 19

Affinity Chromatography

Answer 19

• can also customize columns to bind any protein of interest by creating a column with high affinity for that protein
• accomplished by coating beads with a receptor that binds the protein or a specific antibody to the protein; in either case, the protein is retained in the column.
• Once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody),
  which will compete with the bead-bound receptor and ultimately free the protein from the column.
• The only drawback of the elution step is that the recovered substance can be bound to the eluent.