1.
Biochemistry and molecular biology lab
Lecture 18
Serum protein separation by
cellulose acetate membrane
electrophoresis

2.
Biochemistry and molecular biology lab
Aim
 Learn the principle of cellulose acetate
membrane electrophoresis
 Know the operation and clinical significance of
electrophoresis

3.
Biochemistry and molecular biology lab
Principle
 Electrophoresis:
is the motion of charged particles relative to a
fluid under the influence of an electric field.
Factors determining the electrophoresis motion ：
charge, size, sharp
Based on different supporting materials, there are membrane
electrophoresis and gel electrophoresis
• Gel electrophoresis is the process by which molecules
in a sample can be separated by charge and/or size.

4.
Biochemistry and molecular biology lab
Lab equipments
 electrophoresis device
electrophoresis chamber DYY-2 model power supply
The connection between electrophoresis chamber and power supply

5.
Biochemistry and molecular biology lab
 cellulose acetate membrane
（2 cm×8 cm ）
 Petri dish：staining and rinsing
 Sample applicator (plastic slice)
 Filter paper
 Watch glass
 Forceps

6.
Biochemistry and molecular biology lab
 Rocking table
 722 model
spectrophotometer

7.
Biochemistry and molecular biology lab
Reagents
 fresh serum
 Barbitone sodium Buffer （pH8.6）
 Staining solution（amido black 10B）
 Destaining solution
 0.4mol/L NaOH

8.
Biochemistry and molecular biology lab
Experimental steps
1. Membrane
preparation
Soaking the membrane with
buffer for more than 30
mTinak；e the membrane with forceps, place the
membrane between two pieces of filter paper to
dry the buffer (not too dry), and differentiate
between the smooth and rough surface (rough
surface faces up).
Note：Only touch the margins of the
membrane

9.
Biochemistry and molecular biology lab
2. Sample spotting：
Take a small amount of serum with the plastic slice, and stamp on the membrane
Notes：
Mark with
pencil
 Spotting on the rough surface, 1.5cm from one membrane short
edge
 softly press for 1-2 sec, let the serum absorbed by the membrane
 Spotting only once, no need to repeat
 Spot requirements: thin, straight and not reaching the long edge

10.
Biochemistry and molecular biology lab
3. Place the sample：
Connect the power supply with electrophoresis chamber
Let the rough surface faces down, the spotting side is
placed at the cathode (-) side (Note: do not let the spot
overlap with the supporting paper)
Remove bubbles between the membrane and supporting
paper

11.
Biochemistry and molecular biology lab
4. Electrophoresis ：
 Make sure that the membrane is wet
 Pre-electrophoresis：50V, 5min
 Electrophoresis：Stable voltage, 110V, 40 min

12.
Biochemistry and molecular biology lab
4. Staining and rinsing ：
Staining：Transfer the membrane using forceps to the Amido black 10B
and stain for 3 min
Note：Only touch the margin regions of the membrane with
forceps；Completely submerge every membranes into the staining
solution, no overlapping.
Rinsing：detaining on the rocking table for 3 times
(8 min; 7 min; 6 min)
serum albumin globulins Starting point

13.
Biochemistry and molecular biology lab
5. Quantification：
Cut each bands and a part of the blank membrane, add 4ml
NaOH respectively and shake for 15 min. Determine the light
absorbance at 620 nm.
6. Calculation：
Total absorbance： T= A +α1+ α2+ β+γ
Percentage = ( X/ T ) ×100%
A/G = A/(α1+ α2+ β+γ)

14.
Biochemistry and molecular biology lab
Results
serum albumin
α1 -globulins
α2 -globulins
β- globulins
γ- globulins

15.
Biochemistry and molecular biology lab
Normal range
• serum albumin 57.45-71.73%
• α1-globulins 1.76-4.48%
• α2-globulins 4.04-8.28%
• β-globulins 6.79-11.39%
• γ-globulins 11.85-22.97%
• A/G 1.24-2.36

16.
Biochemistry and molecular biology lab
Clinical significance
 Most of serum protein is produced by liver ，only
γ-globulins produced by plasma cells
 The function of serum proteins ：
Maintain plasma colloid osmotic pressures; maintain
plasm pH balance, base-acid balance; Transport
nutrients, metabolites, hormones, medicines and metal
ions.

17.
Biochemistry and molecular biology lab
 liver cirrhosis：serum albumin, α1, α2 ↓，γ- globulins
↑↑；
 Hepatocarcinoma：between albumin and globulins,
there is an alpha feto protein (AFP) band ；
 acute and chronic nephritis & nephrotic syndrome ：
serum albumin ↓ ， α2 and β globulins ↑；
 Multiple myeloma ： serum albumin ↓ ，γ- globulins
↑ ，between β and γ- globulins, there is a “M” band

18.
Biochemistry and molecular biology lab
Assignment questions
1.Which side (anode or cathode) shall we place
the sample during electrophoresis? Why?
2. What are the possible reasons causing the
irregular, distorted or atypical electrophoresis
bands？

19.
Biochemistry and molecular biology lab
Other electrophoresis techniques ：
1、SDS-Polyacrylamide Gel Electrophoresis
usually used for protein molecular weight
determination

20.
Biochemistry and molecular biology lab
2、Isoelectric focusing electrophoresis：
a technique for separating different molecules by differences in
their isoelectric point (pI)
3、2D electrophoresis：
an important technique in the proteomics
4、Agarose gel electrophoresis：
DNA or RNA separation

21.
Biochemistry and molecular biology lab
Agarose Gel Electrophoresis
Gel electrophoresis is a widely used technique for the
analysis of nucleic acids and proteins. Agarose gel
electrophoresis is routinely used for the preparation and
analysis of DNA.
Gel electrophoresis is a procedure that separates molecules on the basis of
their rate of movement through a gel under the influence of an electrical field.
We will be using agarose gel electrophoresis to determine
the
presence and size of PCR products.

22.
Biochemistry and molecular • DNA is negatively charged. biology lab
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by
size.
- +
Power
DNA
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 μm) 

23.
Biochemistry and molecular biology lab
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
- +
Power
DNA
small
large
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.

24.
Biochemistry and molecular Agar boioslogey lab
D-galactose 3,6-anhydro
L-galactose
•Sweetened agarose gels have
been eaten in the Far East since
the 17th century.
•Agarose was first used in biology
when Robert Koch used it as a
culture medium for Tuberculosis
bacteria in 1882
Agarose is a linear polymer extracted from seaweed.

25.
Biochemistry and molecular biology lab
Making an Agarose Gel
An agarose gel is prepared
by combining agarose
powder and a buffer
solution.
Buffer