1. Last class
Salting out
Dialysis
Paper 1 discussion

2. Purifying proteins Chromatography Mobile phase Stationary phase
Proteins in mobile phase “stick” based on…

3. Gel filtration chromatography

4. Gel filtration chromatography

5. Gel filtration chromatography
Fraction 1

6. Gel filtration chromatography
Fraction 1
Fraction 2

7. Gel filtration chromatography

8. Separating and visualizing proteins
Most proteins – color?
Separate?
Visualize?

9. PAGE

10. PAGE: “Stacking” and “Resolving”
“Stacking” gel
- Lower %age
- “Stacks” samples

11. PAGE: “Stacking” and “Resolving”
“Stacking” gel
- Lower %age
- “Stacks” samples
“Resolving” gel
- Higher %age
- Smaller pores
- Separates proteins

12. 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!

13. SDS PAGE SDS is _____________________ _____________________ proteins
ALL proteins have high –ve charge

14. SDS vs Native PAGE SDS: Separation based on?
Native: Separation based on?
When SDS? When Native?

15. Lab
Gel filtration column
Protein & enzyme assays
PAGE