Electrophoresis and Electroblotting of
Proteins
1
10/11/10
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
2
10/11/10
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
3
10/11/10 In Exercise 6, what was the purpose of DEAE-cellulose in column chromatography?
5
10/11/10
6
4
7
What kinds of protein will end up in a Crude extract?
8
Electrophoresis and Electroblotting of
Proteins
5
10/11/10
9
Metalloproteins
O
2
O
2
O
2
O
2
Hemoglobin
Cytochrome C
Fe++
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10
Channels and Pumps are Membrane Proteins
HYDROPHOBIC
HYDROPHOBIC
Electrophoresis and Electroblotting of
Proteins
6
10/11/10
11
How to keep a Protein
Structure Intact During
Extraction
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12
FOUR LEVELS OF PROTEIN STRUCTURE
PRIMARY:
Ala-Ser-Glut-Val-Tyr-Met-Trp-Phe
SECONDARY
Electrophoresis and Electroblotting of
Proteins
7
10/11/10
13
TERTIARY:
A
B
C
D
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QUATERNARY STRUCTURE:
Electrophoresis and Electroblotting of
Proteins
8
10/11/10
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Extraction Conditions Must Maintain
the Protein in its Native Conformation
and (optimally) Preserve its Activity.
