PP07 - Cells Extracts 1 1 1 1 4 standardcur

PP07 - Cells Extracts 1 1 1 1 4 standardcur

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10/07/10 1 Extraction and Analysis of Proteins From  Cells Preparation of Crude Cell  Preparation of Crude Cell  Extracts Extracts
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10/07/10 2 Things to Learn in This Week’s Lab 1. How to extract proteins from cells. 1. How to make parallel dilutions from a stock solution. 1. How to determine protein concentration using a  standard curve. 4.  How to use the concentration of a dilution to calculate  the concentration of the stock solution.
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10/07/10 3 Extracting Proteins From Cells Why extract proteins from cells? Proteins control virtually all cell activities since they  perform all kinds of functions:    Enzymes Cytoskeletal proteins Metalloproteins  Channels, Pumps G-proteins Motor proteins Receptors Structural Support  Molecules Storage proteins
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10/07/10 4 Proteins are located in all membranes, the cytoplasm, and the interior of all organelles.
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10/07/10 5 Pro te ins  are  am azing ly dive rs e   in terms of: function, structure,  chemical properties charge shape pH optimum size solubility Cofactors This confounds purifying a specific protein.
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6 What kinds of proteinwill end up in aCrude extract?
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10/07/10 7 Enzymes:  globular proteins substrate enzyme Hydrophilic:  Water soluble
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10/07/10 8 Metalloproteins O 2 O 2 O 2 O 2 Hemoglobin Cytochrome C Fe++
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10/07/10 9 Channels and Pumps are Membrane Proteins HYDROPHOBIC HYDROPHOBIC
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10/07/10 10 How to keep a Protein Structure Intact During Extraction
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10/07/10 11 FOUR LEVELS OF PROTEIN STRUCTURE PRIMARY: Ala-Ser-Glut-Val-Tyr-Met-Trp-Phe SECONDARY
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10/07/10 12 TERTIARY: A B C D
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10/07/10 13 QUATERNARY STRUCTURE:
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10/07/10 14 Extraction Conditions Must Maintain the Protein  in its Native Conformation and (optimally)  Preserve its Activity. CARE MUST BE TAKEN TO AVOID DENATURATION
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10/07/10 15 S S Disulfide bond  (strong) (-) C 0= Polypeptide B Polypeptide A HN H NH 3( +) O Hydrogen bond (weak) +/- charge interaction (weak) Polypeptide A
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10/07/10 16 SH =O NH 3 (+) HS H 2 N C O (-) 0= 1.  DTT or B-ME  (reducing agents) 3. Mechanical  Disruption Polypeptide A Polypeptide B Cl(-) Cl(-) Ca++ 2. Change in Ion  Concentration
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10/07/10 17 Effect of pH on Protein Structure and Function
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