Download 1. Electrophoresis of serum protein (CAME)

Experiment Two
1. Electrophoresis of serum
protein (CAME)
2. Assay of casein isoelectric
point ( precipitation method)
2012-09
1. Electrophoresis of serum protein
(CAME)
【Aims】
1. To understand the principle of CAME
( Cellulose Acetate Membrane Electrophoresis )
2. To learn how to analyze the relative conc. of
various proteins in serum
1. Electrophoresis of serum protein
(CAME)
【Principle】
When placed in an electric field, molecules
with a net charge such as proteins, will
move towards one electrode or the other, a
phenomenon known as electrophoresis.
If a protein has more net charges, it will run
faster, otherwise reverse.
According to this phenomenon, the proteins in
serum can be separated into 5 groups,
including A,α1,α2,β,λglobulin
【Principle】
The speed of running depends on the amount of net charge
and molecular weight of the protein molecule
-
+
+
+
+
+
-
+
+
+
+
+
1. Electrophoresis of serum protein
(CAME)
【Protocol】
1. Preparation before Electrophoresis
(1) Dip the piece of CAM in the buffer(pH=8.6)
for ten minutes
(2) Put some serum (2~3µl) on the CAM piece
by pipette
(3) Take the piece of CAM on the
Electrophoresis instrument
(Caution: the side with serum must be put on (-)
electrode)
1. Electrophoresis of serum protein
(CAME)
【Protocol】
2. Electrophoresis
Voltage: about 100~120v
Time: 40~60min
3. Dyeing and rinsing ( washing )
When Electrophoresis finish, take out the piece
and put it into the protein dyeing for 10~15min
then rinsing with the washing solution 1 till its
background is cleaned.
1. Electrophoresis of serum protein
(CAME)
【Protocol】
4. Determination of the amount of proteins
To cut each protein strip and blank strip, put them
into 5ml of the washing solution 2 respectively,
shake up several times, read A500 after half an
hour.
T=A+α1+α2+β+ γ
1. Electrophoresis of serum protein
(CAME)
【Reagents】
1. Barbital buffer (pH8.6)
2. Protein dyeing solution
3. Washing solution1 (C2H5OH 45ml + CH3COOH
5ml + H2O 50ml)
4. Washing solution 2 (0.4mol/L NaOH)
1. Electrophoresis of serum protein
(CAME)
【Caution】
The end of piece of CAM with serum should be
put on the negative polar on the electric field.
Take care about the voltage, don’t leave it too
high which will damage the electric field
because of overheating.
1. Electrophoresis of serum protein
(CAME)
Table 3 Some data of proteins in serum
A
α1
α2
β
γ
pI
4.64
5.06
5.06
5.12
6.85~7.5
Mr (10 thousand )
6.9
5~20
30
9~15
30
57~68
1~6
5~11
7~13
10~18
Albumin
Ratio ％ of
proteins in
serum
1. Electrophoresis of serum protein
(CAME)
【Result】
【Discussion】
2. Assay of casein isoelectric
point ( precipition method)
Aims
To strengthen the comprehension of the
protein ionization and pI
To learn how to assay protein pI
Principle
• When a protein is at its pI, its net charge is
zero and hence its solubility become
minimum.
According to this, we can place casein
in a series of solutions whose pH are
different. By observation of the extent of
the precipitation, the protein pI will be
obtained
R Pr
CH COOH
NH2
R Pr
CH COOH
NH3+
pH<pI
Positively
charged ion
+OH+H+
+OH
R Pr
CH COOR Pr
CH COO-
NH3+
pH=pI
Zwitterion
+H+
NH2
pH>pI
Negatively
charged ion
Protocol
(ml)
Tube No.
1
H2O
2.4
1.00M HAc
1.6
0.10M HAc
2
4.0
3
4
5
3.0
1.5
3.5
2.5
0.5
1.0
0.01M HAc
casein
1.0
1.0
1.0
1.0
1.0
Final pH
3.5
4.1
4.7
5.3
5.9
Precipitation
Observe precipitation, using “+”、 “++” “+++” “++++”
to show the extent of the precipitation
Phenomenon
clear
turbidity
wadding
deposition
turbidity
deposition
clear
deposition
-
+
++
+++
++++