Flashcards in Lecture 8 - Protein Seperation and Purification Deck (29):
Decant off supernatant with soluble proteins
What is added to precipitate proteins
What does salting in means?
At low salt concentrations the solubility of a protein often increases
What does salting out means?
As the concentration of salt increases, the solubility of protein decreases until it precipitates
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
Why do we use ammonium sulfate to precipitate proteins?
Because ammonium sulfate has a larger effect on solubility than other salts
What happens at low salt concentration?
There is a strong attractive force between proteins
What happens at optimal salt concentration?
Salt ions shield charges on proteins so weak attractive force
What happens at high salt concentrations?
Ions compete for water allowing hydrophobic and charge interactions
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
What does chromatography methods exploit?
They exploit the differences in the properties of different proteins
What does chromatographic methods allow?
They allow for the separation of proteins based on some characteristic
Size exclusion chromatography is based on what?
What does ion exchange chromatography based on?
Isoelectric point (charge)
What does affinity chromatography based on?
Interactions with small molecules
In ion exchange chromatography protein mixture is added to column containing what kind of exchangers?
What is a cation exchanger?
It is a negatively charged resin that binds cations
What is an anion exchanger?
It is a positively charged resin that binds anions
True or false? Proteins with smaller chargers elute first. Proteins with the highest charges elute last.
What are the factors that affect ion exchange chromatography?
Net charge on molecule
True or false? In ion exchange chromatography, the smaller the net charge the better the protein binds.
The greater the net charge...
In ion exchange chromatography, what does the pH of the solution affect?
The net charge on a protein
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 or false? In size exclusion chromatography, large molecules can penetrate the pores easily and travel shorter distances - elute at smaller volumes
What happens in affinity chromatography?
Proteins bind to resin via specific interactions with the ligands covalently bound to the resin
Give an example of ligands in affinity chromatography
Antibody that binds protein
What does sodium docecyl sulfate (SDS) used for?
To denature proteins
True or false? There is about 1 SDS molecule for every 4 AA's
2 AA's not 4