Lecture 4

Question 1

What happens at pH 2.5?

Answer 1

a-amino groups exist as NH3+, while a-carboxylate groups exist as 50% COO- (deprot) and 50% COOH (protonated) giving the amino acid an overall positive charge.

Question 2

What does the exact value of overall charge depend on?

Answer 2

Specific pKa values of the various groups in each amino acid.

Question 3

What does the size of net charge determine?

Answer 3

How tightly each amino acid binds.

Question 4

What does high Na+ present in elution buffer first displace?

Answer 4

Weakly bound amino acids

Question 5

What happens as [Na+] is increased?

Answer 5

More tightly bound amino acids are progressively displaced

Question 6

What will an increase in pH lead to?

Answer 6

To eliminate the positive charge on the amino acid, so it no longer binds to then resin.

Question 7

What are proteins derived from?

Answer 7

Microbial cultures, plants or animal tissues such as liver.

Question 8

What is crude extract?

Answer 8

The cells are broken open to release the proteins into a solution.

Question 9

What is separation by ion exchange based on?

Answer 9

Charge differences among proteins.

Question 10

What is charge differences among proteins based on?

Answer 10

The relative number of Asp and Glu (negative) versus His and Lys and Arg (positive) in each protein and on pH

Question 11

What is the percentage of proteins negatively charged at pH 7?

Answer 11

~65%

Question 12

In affinity chromatography, what is the chemical group that is covalently attached to the beads in the column?

Answer 12

Ligand

Question 13

What happens to the proteins that have an affinity towards the ligand?

Answer 13

Bind tightly to it

Question 14

What happens to the other proteins that don’t have an affinity towards the ligand?

Answer 14

Move faster down the column and are eluted from the column.

Question 15

How are the bound proteins eluted?

Answer 15

By the addition of high concentration of salt (weakens the binding between the ligand and the proteins) or ligand (competes with attached ligands)

Question 16

How can an ATP binding protein be isolated?

Answer 16

By attaching a molecule that resembles ATP to the beads.

Question 17

What can Genetic Engineering do?

Answer 17

Fuse a “Tag” to the target protein allowing purification by affinity chromatography.

Question 18

What is a Tag?

Answer 18

A peptide or protein that binds a ligand with high affinity and specifically that is fused to the gene encoding target protein.

Question 19

In metal affinity chromatography, what do clusters of His in a protein bind tightly to?

Answer 19

Ni2+ and Co2+

Question 20

What is a His tag?

Answer 20

6 - 8 extra His residues are fused to the target protein at N- or C- terminus.

Question 21

What is the column made up of?

Answer 21

Chelating resin containing Ni2+

Question 22

What do His-tagged proteins bind to?

Answer 22

Tightly to the Ni2+ resin.

Question 23

How is the bound protein eluted?

Answer 23

Adding imidazole (similar to His side chain) to the buffer which outcompetes His-tag and protein no longer binds to the column.

Question 24

What does high purification in one step cause?

Answer 24

Affect the properties of the protein.

The tag can be removed once the protein is isolated.

Question 25

What does Gel Filtration or Molecular Exclusion chromatography do?

Answer 25

Separates proteins on the basis of size using beads of POLYMERIC GEL - a loose network of polymer with many water filled pores.

• Larger proteins are excluded from the pores
• Protein molecule can enter the pores if they fit.

Question 26

What molecules elute first using Gel Filtration chromatography?

Answer 26

Larger proteins elute first and smaller proteins elute later.

Question 27

What can Gel Filtration be used for?

Answer 27

To measure the molar mass of proteins.

Question 28

What can proteins be separated and characterized by?

Answer 28

Electrophoresis (NOTE: will never use this method to isolate a protein)

Question 29

What can electrophoresis be used to estimate?

Answer 29

• Number of proteins in a mixture
• Degree of purity of a mixture
• Isoelectric Point
• Approximate Molecular Weight

Question 30

In electrophoresis, what does the rate of movement depend on?

Answer 30

Size, shape and change.

Question 31

What polymer is electrophoresis of proteins carried out in gels made up of?

Answer 31

Polyacrylamide: has right porosity for proteins (10kDa - 1000kDa)

Question 32

What are the components of a typical gel?

Answer 32

5 - 15% polymer and 90 - 95% water with conductive buffer.

Question 33

How are separated proteins visualized?

Answer 33

Adding a dye such as Coomassie blue which binds to proteins

Question 34

What does SDS do?

Answer 34

Binds to proteins and partially unfolds them

Question 35

What is SDS?

Answer 35

Sodium dodecyl sulphate, which is a detergent.

Question 36

What do the sulphate moieties of the SDS molecules confer

Answer 36

A large net negative charge on the protein swamping out the native charge

Question 37

All proteins have a:

Answer 37

Uniform charge per size

Question 38

What is separation based strictly on with SDS?

Answer 38

Size

Question 39

In SDS what do smaller proteins do?

Answer 39

Migrate faster than larger ones.

Question 40

What can you do by comparing the positions to which proteins of known molecular weights