Ch. 5 Flashcards
What is protein purification?
Multi-step fractionation process to isolate a single protein from a mixture
What are the steps of cell fractionation?
- Prep cells
- Lyse cells
- Protein activity is retained - Centrifuge to get different fractions
- Separation of organelles - Salting out
- Dialysis
What is salting out?
- Used to exploit differences in solubility of target protein relative to other proteins in the fraction
- Adding increasing amounts of saturated salt solution to protein sample
- Causes formation of insoluble protein aggregates that are functional when resolubilized
- Often use (NH4)2SO4
What is dialysis? (in terms of protein purification)
- Used to remove ammonium sulfate from protein sample
- Uses diffusion to leave protein in buffer of proper ionic strength and pH
- Pores have to be small enough that salt can exit but protein can’t
How should total protein and specific activity change as you purify?
Total protein should decrease as activity increases
What are the different column chromatography techniques?
- Size exclusion/gel filtration
- Ion exchange
- Affinity
What is column chromatography?
Used to separate proteins based on different physical/chemical interactions between the proteins and column matrix
What is size exclusion/gel filtration chromatography?
Separates proteins based on size
What is ion-exchange chromatography?
Exploits charge differences between proteins
What is affinity chromatography?
Exploits specific binding properties of the target protein to separate it from other cellular proteins that lack this binding function
What is gel electrophoresis?
Separates proteins on the basis of charge and size
What is SDS-PAGE?
Separates denatured proteins by size (final step in protein purification)
- Used to approximate molecular mass of protein or determine whether purified protein includes more than 1 polypeptide chain
What do SDS and β-mercaptoethanol do in SDS-PAGE?
- SDS adds a negative charge to all the proteins
- β-mercaptoethanol breaks disulfide bonds
What is isoelectric focusing?
Separates proteins on the basis of charge as a function of pH
What is 2D gel electrophoresis?
Isoelectric focusing combined with SDS-PAGE
- Based on pI and molecular mass