Last class Salting out Dialysis Paper 1 discussion
Purifying proteins Chromatography Mobile phase Stationary phase Proteins in mobile phase “stick” based on…
Gel filtration chromatography
Gel filtration chromatography
Gel filtration chromatography Fraction 1
Gel filtration chromatography Fraction 1 Fraction 2
Gel filtration chromatography
Separating and visualizing proteins Most proteins – color? Separate? Visualize?
PAGE
PAGE: “Stacking” and “Resolving” “Stacking” gel - Lower %age - “Stacks” samples
PAGE: “Stacking” and “Resolving” “Stacking” gel - Lower %age - “Stacks” samples “Resolving” gel - Higher %age - Smaller pores - Separates proteins
DO NOT FOLLOW PROTOCOL IN MANUAL! Staining proteins Stain: Only binds proteins Denville Blue Protein Stain™ Wash off unbound stain DO NOT FOLLOW PROTOCOL IN MANUAL!
SDS PAGE SDS is _____________________ _____________________ proteins ALL proteins have high –ve charge
SDS vs Native PAGE SDS: Separation based on? Native: Separation based on? When SDS? When Native?
Lab Gel filtration column Protein & enzyme assays PAGE