Lecture 6
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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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Lecture 6

Lecture 6

QUESTIONS?

Text 37607

Msg: 118916 Question

QUESTIONS?

Gotohttp://pollev.com/ucibio

Enter question


Lecture 6

Last class

Protein structure

How do proteins fold

Importance of structure


Lecture 6

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?


Lecture 6

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?

  • Solubility

  • Size

  • Charge

  • Affinity/Specificity


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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


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Separating & detecting proteins: PAGE


Lecture 6

Separating & detecting proteins: PAGE


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Separating & detecting: Isoelectric focusing


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