Last class Salting out Dialysis Paper 1 discussion.

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Presentation transcript:

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