Chapter 3.3

Question 1

Describe the purification of proteins starting from the body and its isolation.

Answer 1

1) Homogenization
- >it is the crushing, grinding or blending the tissue of interest into an evenly mixed solution

2) Centrifugation
- >can then isolate proteins from much smaller molecules before other isolation techniques are employed

-the next most common isolation techniques are chromatography and or electrophoresis

Question 2

Describe the process of electrophoresis. Also describe from where to where these compounds travel to.

Answer 2

• works by subjecting the compound to an electric field
• > which then the compounds move according to charge and size
• note negatively charged compounds will migrate towards the positively charged anode
• > positively charged compounds will migrate towards the negatively charged cathode

Question 3

What is the velocity of migration referred to as of a molecule going through electrophoresis?

Answer 3

• it is the migration velocity

- directly proportional to electric field strength and the net charge of the molecule

Question 4

What kind of gel is used for protein electrophoresis?

Answer 4

• polyacrylamide gel is used for protein electrophoresis
• > proteins travel through this matrix in relation to their size and charge
• > smaller particles and highly charged molecules move faster through this electric field

Question 5

Describe the process of Native Page.

Answer 5

-it is a method for analyzing proteins in their native states

Question 6

What are the limitation of native page? What is it useful for?

Answer 6

• it is limited by varying mass to size ratios and mass to charge ratios
• > proteins migrate due to mass to charge ratios
• > different proteins may experience the same level of migration due to these properties

-it is useful to compare the molecular size or charge of the proteins known to be similar in size from other methods

Question 7

Describe the characteristics of SDS-Page? What factors of a compound are taken into account during SDS-Page?

Answer 7

• it is a polyacrylamide gel electropheresis tool
• separates proteins based molecular mass alone
• > add SDS to denature proteins(disrupts noncovalent interactions)
• > SDS binds to proteins and creates large chains with net negative charges

-only the electric field is taken into account and the frictional coefficient(depends on mass) when a protein moves through a SDS-Page gel

Question 8

What is the isoelectric point

Answer 8

• it is the point at which the protein or amino acid is electrically neutral
• > so that means equal number of positive and negative charges
• > for individual amino acids, this is referred to as a zwitterion

Question 9

Describe the process of isoelectric focusing

Answer 9

• it exploits the acidic and basic properties of amino acids
• > separates based on isoelectric point
• > there is an acidic gel at the anode
• > there is a basic gel at the cathode
• > the center is neutral

Question 10

What is the key concept of chromatography

Answer 10

• the more similar the compound is to its surroundings

- >the more it will stick and move slowly through its surroundings

Question 11

When is chromatography preferred over electropherisis?

Answer 11

-it is preferred when large amounts of protein are being separated

Question 12

Describe the process of chromatography

Answer 12

1)place a sample onto the stationary phase or the adsorbent

2) Run the mobile phase through the stationary phase
- >this allows for elution of the compound through the stationary phase or for it to run through(elute just means for the sample to run through the phase)
- >those that have high affinity for the stationary phase will barely migrate at all
- >those that have high affinity for the mobile phase will run much faster

Question 13

What is meant by the term retention time?

Answer 13

-the amount of time a compound spends in the stationary phase

Question 14

Describe the process of column chromatography

Answer 14

• column is filled with silica or alumina beads as an adsorbent
• gravity moves the solvent and compounds down the solvent
• eventually the solvent drips out of the end of the column
• > different fractions that leave the column are collected over time

Question 15

What factors determine how fast a compound moves during column chromatography

Answer 15

1) size
2) polarity
- >less polarity= faster elution

Question 16

Why use column chromatography

Answer 16

• because it can be used to separate and collect macromolecules other that proteins
• > eg; nucleic acids

Question 17

Describe ion-exchange chromatography

Answer 17

• a column is coated with a charged substance
• > they attract or bind to compounds of the opposite charge
• a charged protein is put through the column
• > the column attracts proteins of the opposite charge
• > the oppositely charged proteins will have a longer retention time

Question 18

Describe size-exclusion chromatography

Answer 18

• it contains beads in the column containing tiny pores of varying sizes
• > these tiny pores allow for small compounds to enter, thus slowing them down
• > larger compounds can’t fit into these pores and have to go around the pores
• > so they travel faster

Question 19

Describe affinity chromatography

Answer 19

• columns bind to any protein of interest by creating a column with high affinity for that protein
• > eg; coating the column with receptors that bind to the protein or a specific antibody for that protein
• once the protein is retained on the column
• > it can be washed out with a free receptor(target/antibody)
• > this eluent will compete with the bead-bound receptor and ultimately free the protein from the column

-note eluents can be created with a specific pH or salinity level that disrupts the bonds between ligand and protein of interest