Download + + + - - - Electrophoresis of

Serum protein electrophoresis
Cellulose Acetate Membrane Electrophoresis
(CAE)
xiaoli
What is Electrophoresis ?
Its principle is that the charged particles of
a sample migrate in an applied electrical field.
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anode
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It is applied for the separation and
characterization of proteins, nucleic acids and
subcellular-sized particles like viruses and small
organelles.
Electrophoresis is an analytical method
frequently used in molecular biology and
medicine.
What is function of Electrophoresis ?
Analysis at the level of macromolecules often
involves isolation and purification of a particular one
from among hundreds or thousands, assaying
specifically for this molecule, determining size
(molecular weight), shape, and chemical structure.
Electrophoresis is used to identify and study
charged molecules and macromolecules.
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electrophoretic mobility (v)
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The electrophoretic mobility v of a molecule
is defined as the
migration per unit field strength:
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v = v/x = q / 6πη r
molecule of charge q; electric field of strength x; medium of
viscosityη; a spherical molecule of radius r ; a spherical molecule of
radius r
velocity v
The migration of length per time:
v : cm / min
Migration velocity depends on
Strength of electric field (Heat)
net charge on molecule (pH)
size and shape of molecules (choice of
support)
Properties of supporting medium
(viscosity, electroendosmosis)
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Electrophoresis of
supporting medium
Sodium dodesyl sulphate-plyacrylamide gel
electrophoresis (SDS-PAGE)
Agarose gel electrophoresis
Cellulose acetate membrane electrophoresis
Filter paper
Native gels
Barbitone buffer (pH 8.60, 0.06mol/L)
Albumin
α1-globumin
α2-globumin
β-globumin
γ-globumin
pI=4.9
pI=5.28
pI=5.82
pI=7.3
pI=6.8
Effect of pH and buffer on
protein charge
Proteins are amphoteric compounds
and are therefore either positively or
negatively charged
Isoelectric Point - pH where there is no
net charge in molecule.
NH3+
H- C - COOH
R
PI>pH
H+
NH3+
H- C - COOR
PI=pH
NH2
H- C - COOH+
R
pI<pH
SPECIMENS & MATERIALS
1. Horizontal electrophoresis apparatus
2. Cellulose acetate strips
3. Serum
4. serum applicator
5. Power pack
6. Barbitone buffer (pH 8.60, 0.06mol/L)
7. Whatman 3 MM paper
8. Citrate buffer (pH 6.8, 0.1 mol/L)
9. Amino black 10B stain (0.1%)
10. Washing solution (Methanol: acetic acid: water = 9:1:10)
PROCEDURE
1. Preparation of Apparatus:
Add the buffer into the chambers. Use filter paper
as salt bridge whose one end immerse in the buffer
and another put onto the plastic bracket after the
buffer in the different (anode’s and cathode’s)
chambers have the same height.
2. Prepare the strip of
cellulose acetate
frosting
varnish
b. Immersing the Strips:
Remove the strip of cellulose acetate only with
forceps! On the frosting of the strip, lightly mark a
beeline at 1.5cm of one end with a pencil.
Then moisten a strip by placing it on the
surface of the buffer in a flat dish and allow the
buffer to soak from below. Immerse the strip
completely by gently rocking the dish (about 10 -20
minutes).
3. Sample Applying:
Remove the strip and put between two pieces of
filter paper carefully, obsorbe excess buffer
solution. Fit the applicator to the block and gently
depress, hold down for 3 seconds to completely fill
the slots, and release the applicator slowly.
4. Balancing and Electrophoresis:
Barbitone buffer (pH 8.60, 0.06mol/L)
Albumin
α1-globumin
α2-globumin
β-globumin
γ-globumin
pI=4.9
pI=5.28
pI=5.82
pI=6.8
pI=7.3
5. Staining:
When electrophoresis is completed, turn off the
power and disconnect the cords BEFORE removing
the chamber cover.
Remove the strips, and stain with for 5-10 min in
amino black 10 B.
6. Washing and Observing:
Excessive dye from the strip is released by
washing repeatedly.
Rinse in washing solution for three separate
rinses of at least 2 min each with mild agitation.
Strips may be stored in 5% acetic acid in the
refrigerator if necessary. Strips may be preserved
for a report by blotting, air drying and taping to a
report form with transparent tape.
Washing solution (Methanol: acetic acid: water = 9:1:10)
1=albumin；
2，3，4，5 is α1-，α2-，β- andγ-globumin；
6 is origine
Clinic significance
(a)
A a1 a2
(b)
b
g
1. Normal value:
A 62％～71％
After scanning
a1 3％～4％
a2 6％～10％
A a1 a2
b
g
b
7％～11％
g
9％～18％
hepatocirrhosis
Normal
hepatocirrhosis
Normal
nephrotic syndrome
medullary tumour
medullary tumour
disease
albumin
α1globulin
α2
β
γ
Kidney disease
↓↓
↑
↑↑
↑
↓
Diffuse liver damage
↓↓
↑
↓
↓
↑
hepatocirrhosis
↓↓
↓
↓
Idiopathic liver cancer
↓↓
AFP
↑
medullary tumour
↑
Chronically
inflammation
↓
gestation
↓
No- γglobulindisease
Double albumin
disease
β-γbridge
↑
↑
↑↑
↑
↑
↓
↓↓
Double apex
Acidic
and Basic
amino
acid