Electrophoresis and Electroblotting of Proteins 110/11/10 1 Extraction and Analysis of Proteins From Cells 10/11/10 2 Things to Learn in This Week’s Lab 1.How to extract proteins from cells. 2.How to make parallel dilutions from a stock solution. 4.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.
Electrophoresis and Electroblotting of Proteins 210/11/10 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 10/11/10 4 Proteins are located in all membranes, the cytoplasm, and the interior of all organelles.
Electrophoresis and Electroblotting of Proteins 310/11/10 In Exercise 6, what was the purpose of DEAE-cellulose in column chromatography?5 10/11/10 6
47 What kinds of protein will end up in a Crude extract? 8
Electrophoresis and Electroblotting of Proteins 510/11/10 9 Metalloproteins O2 O2 O2 O2 Hemoglobin Cytochrome C Fe++ 10/11/10 10 Channels and Pumps are Membrane Proteins HYDROPHOBIC HYDROPHOBIC
Electrophoresis and Electroblotting of Proteins 610/11/10 11 How to keep a Protein Structure Intact During Extraction 10/11/10 12 FOUR LEVELS OF PROTEIN STRUCTURE PRIMARY: Ala-Ser-Glut-Val-Tyr-Met-Trp-Phe SECONDARY
Electrophoresis and Electroblotting of Proteins 710/11/10 13 TERTIARY: A B C D 10/11/10 14 QUATERNARY STRUCTURE:
Electrophoresis and Electroblotting of Proteins 810/11/10 15 Extraction Conditions Must Maintain the Protein in its Native Conformation and (optimally) Preserve its Activity.