1. Lecture 7 Web: pollev.com/ucibio Text: To: 37607 Type in: 169964

2. The importance of structure “Chaperones” & protein folding Misfolded proteins are very, very bad! E.g. Huntington’s disease E.g. Prions

3. Aggregation of mutant Huntington’s “Tag” protein - GFP Put “tagged” proteins in cells Compare: - Normal - Mutant

4. Prions are WHACK proteins! Normal vs Disease = Same gene (no mutations!) Put Prion version into normal cells Prion  Normal = Diseased! Prion protein changes 3D conformation

5. Working with proteins Interested in studying Hexokinase How many proteins in cell? First step in studying Hexokinase? How do you know when you have purified Hexokinase?

6. Estimating purity First step – how do we know our protein is present? How do we know how pure our protein is?

7. Estimating purity Assume 100 proteins in cell. 1g of each protein. Total protein? Ratio of our protein? Purify: Get rid of 50 unwanted proteins. Total protein? Ratio of our protein? Purify: Get rid of 40 remaining unwanted proteins. Total protein? Ratio of our protein? Purify: Get rid of 8 remaining unwanted proteins. Total protein? Ratio of our protein? Ratio of activity of protein:Total protein = Specific activity

8. Specific activity table

9. Purifying proteins 1 st step = Getting protein! - Lyse - “Fractionate”

10. Cell fractionation by centrifugation

11. Purifying proteins What properties of proteins can you exploit?

12. Changing protein solubility – Salting in/out http://www.foodnetworksolution.com/wiki/word/1820/salting-out

13. Changing protein solubility: pH & Charge http://elte.prompt.hu/sites/default/files/tananyagok/practical_biochemistry/ch05s04.html

14. Column chromatography: General principle

15. Gel filtration column: Size

16. Ion exchange column: Charge

17. Affinity column

18. Immunoprecipitation