Lecture 8 - Protein Seperation and Purification Flashcards Preview

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Flashcards in Lecture 8 - Protein Seperation and Purification Deck (29):
0

Decant off supernatant with soluble proteins

Soluble lysate

1

What is added to precipitate proteins

Ammonium sulfate

2

What does salting in means?

At low salt concentrations the solubility of a protein often increases

3

What does salting out means?

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

4

What are the steps of ammonium sulfate precipitation procedure?

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

5

Why do we use ammonium sulfate to precipitate proteins?

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

6

What happens at low salt concentration?

There is a strong attractive force between proteins

7

What happens at optimal salt concentration?

Salt ions shield charges on proteins so weak attractive force

8

What happens at high salt concentrations?

Ions compete for water allowing hydrophobic and charge interactions

9

What happens in column chromatography?

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

10

What does chromatography methods exploit?

They exploit the differences in the properties of different proteins

11

What does chromatographic methods allow?

They allow for the separation of proteins based on some characteristic

12

Size exclusion chromatography is based on what?

Size/shape

13

What does ion exchange chromatography based on?

Isoelectric point (charge)

14

What does affinity chromatography based on?

Interactions with small molecules

15

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

Cation exchangers

16

What is a cation exchanger?

It is a negatively charged resin that binds cations

17

What is an anion exchanger?

It is a positively charged resin that binds anions

18

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

True

19

What are the factors that affect ion exchange chromatography?

Net charge on molecule

Solution pH

Ionic strength

20

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

False.
The greater the net charge...

21

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

The net charge on a protein

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

True

23

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

False
CANNOT

24

What happens in affinity chromatography?

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

25

Give an example of ligands in affinity chromatography

Co-enzyme
Inhibitor
Antibody that binds protein

26

What does sodium docecyl sulfate (SDS) used for?

To denature proteins

27

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

False
2 AA's not 4

28

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

Negatively