生物化学与分子生物学实验 experiment six cellulose acetate film electrophoresis separate...

生物化学与分子生物学实验

Experiment Six

Cellulose acetate film electrophoresis

separate serum proteins


Purpose

learning the main procedure and clinical

significance of cellulose acetate film;

grasp the principle of cellulose acetate

film;


Principle Electrophoresis: Suspended particles have an electric surface

charge, strongly affected by surface adsorbed species, on

which an external electric field exerts

an electrostatic Coulomb force. molecules are separated by

applying an electric field to move the negatively charged

molecules through a matrix of agarose or other substances

Effectors of electrophoresis:

electric charge 、 quantity 、 molecular size 、 shape

Categories: film electrophoresis 、 paper electrophoresis, agarose electrophoresis

http://en.wikipedia.org/wiki/Electrophoresis


PI of serum protein is range 4.0-7.3, and in

the pH 8.6, all of protein contain negative

charge and move to positive electrode.

different proteins contain

different PI value, different

molecular weight; the smaller of

the protein, the quicker they

move.


Apparatus

DYY-2 mode electrophoresisHorizontal electrophoresis tank

Connection of tank and power

Electrophoresis devise


cellulose acetate film (2

cm×8cm) ： 1 piece /person

culture dish: all 4

sets ， including

dyeing （ 1 ）， washing

（ 3 ） point sampler

filter paper

surface dish

pincette/nipper


decolorized shaker

722 Uv-vis spectrophotometer


Reagent

fresh human serum

babital buffer (pH8.6)

dyeing solution （ acid blue black 10b ）

washing solution

0.4mol/L NaOH (sodium hydroxide)


1. Film preparation

Procedure

Put the film in barbital solution, and sink for at least 30min;

Use nipper to remove soaked film, put it in two-piece filter paper to remove extra water(could not be too dry);then flat out on the paper (rough side upwards);


Using sample applicator picking up a minute quantity of serum, spotting as stamp method

2. Sample application ：

spotting on the rough side, 1.5cm from one edge and at the center. pressing for 1-2s, make sure the serum penetate inside the film. spotting only once; the line should be thin, straight and do not touch the edge.

Notice ：


According to the size of the tank, make the proper size filter paper bridge;

3. Upload sample ：Connecting tank and power;

Put film across the bridge with rough side downwards; and with the sample side near negative charge; (notice: sample point could not touch the bridge )

Exclude bulb of the film


Balance: close tank cover, balancing 5min to wet film

completely;

Pre-electrphoresis:50V, 5min;

Electrophoresis: stable voltage at 120V for 1h(the first

line reach 2/3 of the whole film).

4. Electrophoresis:


dyeing: using nipper to remove film to acid blue black 10b

solution for 1-3min;

notice: nipper can only catch out side of the film; each film need to

be completely sunk;

5. Dyeing and decolorizing

decolorizing: put on the shaker to decolorize at 5min per time and for 3-4 times;


6. Quantity determination ：

cut each district and margin film, adding 4 ml NaOH

separately for 15-20min(shaking at the same time), then

detecting absorption at 620nm;7. Calculation:

absorption sum of each district: T=A+α1+ α2+ β+γ

relative content= ( X / T ) ×100%

A / G = A / (α1+ α2+ β+γ)


Result

albumin

α1- globulin

α2 -globulin

β-globulin

γ-globulin


• albumin 57.45~71.73%

• α1 -globulin 1.76~4.48%

• α2 –globulin 4.04~8.28%

• β-globulin 6.79~11.39%

• γ-globulin 11.85~22.97%

• A/G 1.24~2.36

Normal reference


Clinical significance Most of Plasma proteins are synthesized by liver, and

only γ-globulin is synthesized by plasma cell;

Function of plasma protein:

maintaining the osmotic pressure of plasma; including

transport of lipids, hormones, vitamins and metals in the

circulatory system; the regulation of cellular activity

and functioning and in the immune system.

http://en.wikipedia.org/wiki/Blood_proteins


Cirrhosis：清蛋白、 α1 、 α2↓ ， γ-球蛋白↑↑；

HCC (Hepatocellular carcinoma) ：清蛋白与球蛋白间多出一条甲胎蛋白（ AFP）带；

acute/chronic nephritis ：清蛋白↓， α2 和

β球蛋白↑； multiple myeloma：清蛋白↓ ， γ-球蛋白↑ ，β和 γ-球蛋白区带之间出现“ M”带

Disease:


1.电泳时，血清样品点在支持介质的哪一端，为什么？

2. 引起电泳图谱不整齐的原因有哪些？

Questions


其他主要电泳技术介绍：

1 、 SDS- 聚丙烯酰胺凝胶电泳常用于蛋白质分子量的测定，有垂直板、平板、

盘状电泳


2 、等电聚焦电泳：通过蛋白质等电点的差异而分离蛋白质。3 、双向凝胶电泳：蛋白质组学研究的重要技术。

4 、琼脂糖凝胶电泳：分离 DNA 或 RNA 分子

生物化学与分子生物学实验 experiment six cellulose acetate film electrophoresis separate...

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