Characterizing Erythrocyte Membrane Proteins by SDSPAGE Part 2

1
Characterizing Erythrocyte Membrane Proteins by
SDS-PAGE Part 2
2
Macromolecules and Molecular Mass
G-actin
45,000 gms
ONE MOLE
Molecule of NaCl
58 gms
Molecule images are not to the same scale
3
Protein Structure
Portion of polypeptide sequence
Gly-Ile-Val-Glu-Gln-Cys-Cys-Ala-
Alpha helix (blob view)
Portion of folded polypeptide
Functional insulin molecule
4
Secondary and Tertiary Structures
3-dimensional structure of ß-hemoglobin
Alpha-helical segment
5
Quaternary Structure
Native hemoglobin
Alpha subunit
Heme
Beta subunit
6
Fundamental Forces Shape3-D Structure
Hydrogen bonds in alpha helix
Micelle
head (hydrophilic) tail (hydrophobic)
Single hydrogen bond (dashed line)
Phospholipid
7
SDS Takes Proteins Apart
Native proteins

Sodium dodecyl sulfate
Denatured polypeptides
8
Reducing Agent Eliminates Disulfide Bonds
cysteines
Reducing conditions
single polypeptides
dimer
9
SDS-Polyacrylamide Gels
effective separation
poor separation
Liquid or very porous gel
Polyacrylamide matrix w/SDS
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Slab Gel Design
Comb, a.k.a. well former (removed)
Sample well
Stacking gel (pH 7)
Separating gel (pH 9)
Back plate
Spacer
Front plate
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Setting up a Vertical Gel
Cathode
Upper buffer compartment
Gel cassette
Lower buffer compartment
Anode
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Loading a Sample
ELECTRODE BUFFER
Sample in syringe
portion of stacking gel
lane divider
thickness is exaggerated
13
Starting the Run
Cathode (-)
glycine
Electrode buffer
proteins and tracking dye
Cl-
Anode ()
14
Electric Current
R
V ()
(-)
v1
i
R
2V ()
(-)
v2
2i
15
Ionic Conduction
i
A
V ()
1M NaCl
(-)
2i
B
2V ()
(-)
1M NaCl
sodium ion
chloride ion
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Ionic ConcentrationAffects Ion Mobility
i
A
1M NaCl
(-)
()
10 cm/hr
i
B
(-)
()
0.5M NaCl
sodium ion
20 cm/hr
chloride ion
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Electrophoretic Mobility
sodium ion
chloride ion
i
1M
0.1M
1M
(-)
()
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Moving boundaries
Cathode (-)
Electrode buffer
glycine
proteins and tracking dye
region of high mobility
Cl-
Anode ()
19
Stacking Effect
Lane dividers cut off for clarity
protein bands
glycine
dye
Stacking gel, pH 7
pH change
Separating gel, pH 9
Cl-
20
Band Separation
Stained gels
7 T
10 T
12 T
Top of gel
A
A albumin
A
Same high molecular mass proteins
A
Dye front
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Gel Density, Resolution, and Calibration
7 gel

• Low density gel (e.g., 7) resolves heavier
  proteins
  
• High density gel (e.g., 14) resolves lighter
  proteins
  
• MW standards calibrate each gel

Calibration standards
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Teamwork

• Partner with another team
• Prepare one type gel (7 or 14)
• Other team prepares the other type gel
• Load both sets of samples on each gel
• Use both gels for analysis
• Choose best samples for analysis