Protein purification and characterisation Flashcards
What are the advantages of using bacteria to overexpress interesting genes?
- very easy to manipulate
- easy to grow in large quantities
- potentially very high yields of protein
What are the disadvantages of using bacteria to overexpress interesting genes?
- post-translational modifications will probably be different to those used in eukaryotic cells
- Poor at folding complicated proteins
What are the advantages of using unicellular eukaryotes to overexpress interesting genes?
- easy to genetically manipulate
- easy to grow in large quantities
- potentially high yields of protein
- post-translational modifications more mammalian ones
What are the disadvantages of using unicellular organisms to overexpress interesting genes?
- only moderate ability fold complicated proteins
What are the advantages of using mammalian cells to overexpress interesting genes?
- Full range of mammalian post-translational modifications
- Ability to fold and assemble complicated proteins
What are the disadvantages of using mammlian cells to overexpress interesting genes?
- Less easy to genetically manipulate
- hard to grow in large quantities
- poor yields of protein
- very expensive growth media
How can you find out if a protein is present in a sample?
Test for activity by doing an assay
What is the most common technique for purifying large quantities of protein?
Column chromatography
What is the stationary phase in column chromatography?
The porous solid matrix (the gel)
What are the 4 types of column chromatography?
- Gel filtration
- Ion exchange
- Hydrophobic interaction
- Affinity Chromatography
What does affinity chromatography separate proteins on?
The basis of specific protein interactions e.g. enzyme binding to a substrate
What does gel filtration chromatography separate proteins based on?
The size of the proteins
What is the isoelectric point of a protein?
The pH at which a protein has no overall charge
What is the charge of a protein at a pH greater than its Pi?
Negative
What is the charge of a protein at a pH lower than its Pi?
Positive
How can the charge of a protein be changed?
Changing the pH of the buffer
Give 2 examples of common ion exchange chromatography media
- DEAE cellulose
- CM cellulose
What is the charge of DEAE cellulose media?
Positive
What is the charge of CM cellulose media?
Negative
What are the 2 ways we can make a protein unbind from a column?
- Change the pH of the buffer flowing through the column to change the charge on the protein
- Increase the buffer [salt] to elute the protein via charge shielding
What is used to determine the purity of a protein?
SDS-PAGE
