Purification & Seperation of Proteins Flashcards
Purification: Step 1
Centrifugation
-Break open tissues or cell
-CRUDE EXTRACT
Purification: Step 2
Fractionation
-Separate proteins –> Fractions
-Fractions based on SIZE or CHARGE
-“Salting out” = Lower solubility of proteins in SALT –> Precipitates
Purification: Step 3
Dialysis
-Semi-permeable membrane = separates proteins from small solutes.
Column Chromatography
Step 1: Buffer solution (MP) migrates through porous beads (solid phase)
Step 2: Buffer solutions that contain proteins migrate through the solid phase (Elute or Elusion)
Protein properties determine how fast a protein migrates through
Ion Exchange Chromatography
-Based on sign & magnitude of the net electric charge
2 Types of Bound Charge Groups:
-Cation Exchanges
-Anion Exchange
Size-Exclusion Chromatography (Gel Filtration Chromatography)
-Based on size
-Larger proteins -> Exit faster
-Smaller proteins –>Entrapped by porous beads
Affinity Chromatography
-Based on the binding of affinity
-Eluted by High concentrations of salt or ligand.
High-Performance Liquid Chromatography
-High-pressure pumps are used to move proteins down the column.
-IMPROVES RESOLUTInON (More direct protein)
Electrophoresis
Visualize and characterize purified proteins.
Proteins separated by Molar Mass
Polyacrylamide Gels Electrophoresis (PAGE)
The protein migrates based on the charge-to-mass ratio
-Visualization: Coomassie blue dye- Binds to proteins.
Sodium Dodecyl Sulfate (SDS)
Def= A detergent.
Function:
-Binds and partially unfolds proteins.
-Gives proteins all similar charge-to-mass ratio
-Separates proteins by Molecular weight.(Smaller -> Faster)
PAGE & SDS
Due to the partially unfolded proteins and allowing all proteins to have similar charge-to-mass ratio. In which indicated on PAGE smaller proteins migrate faster down the gel.
