Lecture 12 - Protein Purification Flashcards
1
Q
Protease Inhibitors
A
- Used to block degradation of target enzyme during its isolation from the cell
- Usually immediately after cell breakage when cellular proteases are released along with target protein
- Can be made or purchased
2
Q
Protein Purification Procedures
A
- Vary depending on Solubility, Ionic Charge, Polarity, Size, and Binding Specificity
3
Q
Centrifugation
A
- Rotors that spin to get rid of insoluble biomolecules and unbroken cells
- Used to isolate purified protein
4
Q
Solubility - “Salting In”
A
- The observation that protein solubility increases with increasing salt concentration
- Counterions from salt shield protein molecules’ ionic charges, less aggregation
- Proteins least soluble at pI of protein
- Occurs in lower salt concentration range (0-250 mM)
5
Q
Solubility - “Salting Out”
A
- The observation that protein solubility decreases with increasing salt
- Occurs with large amount of salt, up to about 4 M
- Water solvates the salt ions, protein molecules forced together
- Hydrophobic interactions become more important and proteins precipitate
6
Q
Fractionation by Salting Out
A
- Add salt, some proteins will precipitate out, centrifuge, than add more salt
- Repeat process until precipitate displays target biological activity when dissolved again
7
Q
Ion Exchange Chromatography - Cation Exchange
A
- Resins are negatively charged, bind positive ions
- Uncharged or negatively charged ions pass through
- Very useful with large volumes of protein
8
Q
A
9
Q
Ion Exchange Chromatography - Anion Exchange
A
- Resins are positively charged, bind negative ions
- Uncharged or positively charged ions pass through
- Very useful with large volumes of protein
10
Q
Size-Exclusion/Gel Filtration/Gel Permeation Chromatography
A
- Resin beads have pores within them, can separate proteins based on size
- Also used to determine the quaternary structure of a protein
- “Void volume” is the volume of solvent space surrounding the beads, volume of buffer needed to elute the largest proteins
- “Bed volume” is the total volume of the column, volume of buffer needed to elute the smallest proteins
- “Elution volume” is the volume of solvent needed to elute the specific protein
11
Q
Affinity Chromatography
A
- Uses a specialized resin, chemically attached to a ligand, to which your protein will specifically bind
- Protein mixture is run through the column and the protein of interest will bind, everything else washed out
- Can be used with fairly large volumes of protein
12
Q
Dialysis
A
- Technique to dilute high salt concentrations, separate small molecules from larger ones
- Change buffer concentration
13
Q
Isoelectric Focusing (IEF)
A
- Separates on the basis of pI
- Proteins migrate in a pH gradient parallel to electric field
- HCL at + electrode and NaOH at -electrode
- Can isolate once each protein is in correct region
