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Lecture 6

Lecture 6. QUESTIONS? Text 37607 Msg : 118916 Question. QUESTIONS? Goto http://pollev.com/ucibio Enter question. Last class. Protein structure. How do proteins fold. Importance of structure. Working with proteins. Interested in studying Hexokinase. How many proteins in cell?.

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Lecture 6

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  1. Lecture 6 QUESTIONS? Text 37607 Msg: 118916 Question QUESTIONS? Gotohttp://pollev.com/ucibio Enter question

  2. Last class Protein structure How do proteins fold Importance of structure

  3. 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?

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

  5. 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

  6. Specific activity table

  7. Purifying proteins 1st step = Getting protein! • Lyse • “Fractionate”

  8. Cell fractionation by centrifugation

  9. Purifying proteins What properties of proteins can you exploit? • Solubility • Size • Charge • Affinity/Specificity

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

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

  12. Column chromatography: General principle

  13. Gel filtration column: Size

  14. Ion exchange column: Charge

  15. Affinity column

  16. Immunoprecipitation

  17. Immunoprecipitation

  18. Separating & detecting proteins: PAGE

  19. Separating & detecting proteins: PAGE

  20. Separating & detecting: Isoelectric focusing

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