lecture 7 web: pollev.com/ucibio text: to: 37607 type in: 169964
TRANSCRIPT
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Lecture 7
Web: pollev.com/ucibio
Text: To: 37607Type in: 169964 <your
question>
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The importance of structure
“Chaperones” & protein foldingMisfolded proteins are very, very bad!E.g. Huntington’s disease
E.g. Prions
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Aggregation of mutant Huntington’s
“Tag” protein - GFP
Put “tagged” proteins in cellsCompare: - Normal - Mutant
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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
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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?
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Estimating purity
First step – how do we know our protein is present?
How do we know how pure our protein is?
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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
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Specific activity table
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Purifying proteins
1st step = Getting protein!
- Lyse
- “Fractionate”
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Cell fractionation by centrifugation
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Purifying proteins
What properties of proteins can you exploit?
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Changing protein solubility – Salting in/out
http://www.foodnetworksolution.com/wiki/word/1820/salting-out
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Changing protein solubility: pH & Charge
http://elte.prompt.hu/sites/default/files/tananyagok/practical_biochemistry/ch05s04.html
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Column chromatography: General principle
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Gel filtration column: Size
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Ion exchange column: Charge
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Affinity column
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Immunoprecipitation
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Immunoprecipitation