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Slide1
Lecture 7
Web: pollev.com/ucibio
Text:
To: 37607
Type in: 169964 <your question>Slide2
The importance of structure
“Chaperones” & protein folding
Misfolded
proteins are very, very bad!
E.g. Huntington’s disease
E.g. PrionsSlide3
Aggregation of mutant Huntington’s
“Tag” protein
- GFP
Put “tagged” proteins in cells
Compare:
- Normal
- MutantSlide4
Prions are WHACK proteins!
Normal
vs
Disease = Same gene (no mutations!)
Put Prion version into normal cells
Prion
Normal = Diseased!
Prion protein changes 3D conformationSlide5
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?Slide6
Estimating purity
First step – how do we know our protein is present?
How do we know how pure our protein is?Slide7
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 activitySlide8
Specific activity tableSlide9
Purifying proteins
1
st
step = Getting protein!
Lyse
“Fractionate”Slide10
Cell fractionation by centrifugationSlide11
Purifying proteins
What properties of proteins can you exploit?Slide12
Changing protein solubility – Salting in/out
http://www.foodnetworksolution.com/wiki/word/1820/salting-outSlide13
Changing protein solubility: pH & Charge
http://elte.prompt.hu/sites/default/files/tananyagok/practical_biochemistry/ch05s04.htmlSlide14
Column chromatography: General principleSlide15
Gel filtration column: SizeSlide16
Ion exchange column: ChargeSlide17
Affinity columnSlide18
ImmunoprecipitationSlide19
Immunoprecipitation