1.

Voltage power supply

2. Electrophoresis tank
3. 0.5M Barbitone buffer pH 8.2 & 8.6
4. Mixture of both buffer at 1:1 ratio
5. 0.2% Ponceau S
6. 5% Acetic acid
7. Filter paper
8. Cellulose acetate strip
9. Forceps
10. Magnet
11. 0.5ml serum sample
12. Capillary tube

1. measure 250ml Barbitone buffer pH8.2

2. pour buffer into cathode compartment
3. measure 250ml Barbitone buffer pH 8.6
4. pour into anode compartment
5. using forceps take piece of filter paper and soak in buffer pH8.2,
place over the bridge of cathode compartment.
6. take another, soak in pH8.6, place over bridge of anode
compartment
7. measure 250 ml mixed buffer, pour into a container compartment
8. soak CAS in mixed buffer for abt 3 mins
9. remove and dry by placing in between 2 piece of filter paper
10. place CAS across 2 electrode. make sure 0.5-1cm of end of strip
is placed above filter paper
11. use magnet to ensure CAS is flat and taut.
_

12. cover tank and pass electric current at 200v

13. allow strip to equilibrate for 10min
14. switch off power, remove tank cover
15. use capillary tube apply 10 microlitre of serum smple as a
abt 1 cm from cathode end.
16. cover tank and pass current at 200v for 19min
17. switch off and remove tank as well as magnet
18. use forceps to remove CAS
19. soak CAS in ponseau for 10 mins
20. pour AA in container
21. remove CAS and rinse in AA until all excess colouring agent
removed
22. repeat rinse to achieve clearer background
23. dry at room temp
24. separate protein bands are visible
 -
top → bottom ×
left → right ✗

① cathode → anode ④✓

•* +

if ⑧ +
I

of protein
amt &
intensity: thickness

-
 BIOCHEMISTRY DEPARTMENT
FACULTY OF MEDICINE
UNIVERSITY KEBANGSAAN MALAYSIA

ELECTROPHORETIC SEPARATION OF SERUM PROTEIN (ANSWER SCHEME)

1.
1.1 A: Albumin
B: α1
C: α2
D: β
E: γ

1.2 Principle of serum proteins electrophoresis:

Charge particles under the influence of an electric field will migrate to the
electrode of the opposite charge. Positive ions (cations) will migrate to the
cathode, the negative electrode. Negative ions (anions) will migrate to the
anode, the positive electrode.

1.3 Because in alkaline pH protein/amino acid will be negatively (–ve) charged. This
will make protein move from cathode (-ve) (like-like charges repel) to anode
(+ve).

1.4 Examples of plasma proteins:

Bands Plasma proteins Diseases/Conditions

Albumin Albumin Hypoalbuminaemia in malnutrition, nepthrotic
syndrome and chronic liver disease such as
liver cirrhosis, liver cancer.
α1 α1-antitrypsin (AAT) Emphysema: Low in AAT, enzymes produced
by the lungs in response to infections and
toxins (such as cigarette smoke) will slowly
destroy the lung tissue
α2 Caeruloplasmin Wilson’s disease: caeruloplsamin is low which
is an inborn error of copper metabolism, liver
cannot synthesize caeruloplasmin that cause
deposition of copper in the liver  liver
cirrhosis
β Ferritin Reduced in iron deficiency anemia
Increased in haemochromatosis along with
serum Fe (genetic disorder, accumulation of
iron in body tissues causing organ
dysfunction. Eg. hepatomagaly)

1
 Low density Increased level indicates high cholesterol in
lipoprotein (LDL) blood that will lead to artherosclerosis
γ Immunoglobulin IgG, Low immunoglobulins in serum due to
IgM, IgD Haematological disorder eg. myeloma,
chronic lymphatic leukemia
High during infection

2.
2.1 γ-globulin is high in this serum electrophoregram.
Example of diseases:
chronic infections, chronic liver disease
Autoimmune disease
Paraproteinaemia eg multiple myeloma
3.
3.1 3 Causes of hypoalbunaemia:
Malnutrition, malabsorption
Nephrotic syndrome (kidney disease)
Liver disease

3.2 Hypoalbuminemia:
Function of albumin is to maintain oncotic pressure, thus hypoalbuminemia will
cause low intravascular osmotic pressure (in normal condition, hydrostatic =
oncotic pressure). This condition makes fluid enters the extracellular space
(interstitial) causing edema since hydrostatic pressure > osmotic pressure.