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