Lecture 8 - Protein Seperation and Purification

Question 0

What is added to precipitate proteins

Answer 0

Ammonium sulfate

Question 1

Decant off supernatant with soluble proteins

Answer 1

Soluble lysate

Question 2

What does salting in means?

Answer 2

At low salt concentrations the solubility of a protein often increases

Question 3

What does salting out means?

Answer 3

As the concentration of salt increases, the solubility of protein decreases until it precipitates

Question 4

What are the steps of ammonium sulfate precipitation procedure?

Answer 4

1. Precipitate in the mood undesired proteins by adding low salt concentration
2. Precipitated and isolate desire protein by increasing the salt concentration

Question 5

Why do we use ammonium sulfate to precipitate proteins?

Answer 5

Because ammonium sulfate has a larger effect on solubility than other salts

Question 6

What happens at low salt concentration?

Answer 6

There is a strong attractive force between proteins

Question 7

What happens at optimal salt concentration?

Answer 7

Salt ions shield charges on proteins so weak attractive force

Question 8

What happens at high salt concentrations?

Answer 8

Ions compete for water allowing hydrophobic and charge interactions

Question 9

What happens in column chromatography?

Answer 9

1. Protein solution is loaded onto a call impact with the hydrated porous matrix
2. Buffer is pumped through the column
3. Proteins are separated by some property
4. Effluent is collected in fractions and each fraction contains a different proteins or mixture of proteins

Question 10

What does chromatography methods exploit?

Answer 10

They exploit the differences in the properties of different proteins

Question 11

What does chromatographic methods allow?

Answer 11

They allow for the separation of proteins based on some characteristic

Question 12

Size exclusion chromatography is based on what?

Answer 12

Size/shape

Question 13

What does ion exchange chromatography based on?

Answer 13

Isoelectric point (charge)

Question 14

What does affinity chromatography based on?

Answer 14

Interactions with small molecules

Question 15

In ion exchange chromatography protein mixture is added to column containing what kind of exchangers?

Answer 15

Cation exchangers

Question 16

What is a cation exchanger?

Answer 16

It is a negatively charged resin that binds cations

Question 17

What is an anion exchanger?

Answer 17

It is a positively charged resin that binds anions

Question 18

True or false? Proteins with smaller chargers elute first. Proteins with the highest charges elute last.

Answer 18

True

Question 19

What are the factors that affect ion exchange chromatography?

Answer 19

Net charge on molecule

Solution pH

Ionic strength

Question 20

True or false? In ion exchange chromatography, the smaller the net charge the better the protein binds.

Answer 20

False.

The greater the net charge…

Question 21

In ion exchange chromatography, what does the pH of the solution affect?

Answer 21

The net charge on a protein

Question 22

True or false? In size exclusion chromatography, small proteins can diffuse in and out of the pores of the resin increasing the distance traveled through column - require larger volumes of buffer to elute

Answer 22

True

Question 23

True or false? In size exclusion chromatography, large molecules can penetrate the pores easily and travel shorter distances - elute at smaller volumes

Answer 23

False

CANNOT

Question 24

What happens in affinity chromatography?

Answer 24

Proteins bind to resin via specific interactions with the ligands covalently bound to the resin

Question 25

Give an example of ligands in affinity chromatography

Answer 25

Co-enzyme
Inhibitor
Antibody that binds protein

Question 26

What does sodium docecyl sulfate (SDS) used for?

Answer 26

To denature proteins

Question 27

True or false? There is about 1 SDS molecule for every 4 AA’s

Answer 27

False

2 AA’s not 4

Question 28

Each SDS molecule is _________ charged so that he SDS charge overwhelms the protein charge and separation is dependent on mass

Answer 28

Negatively