Practical courses of Biochemistry and Molecular biology

Think ?
Why to do ?

How to do?

Requirements :
1. Do experiment seriously . 2. Write down your results carefully.

3. Submit your report on time.

4. Do cleaning work.

Basic techniques for biochemistry

1. Spectrophotometry: 2. Chromatography:

3. Electrophoresis:
4. Centrifugation:

Experiment 1
Electrophoresis Technology

Question :

You are given a plastic tube containing small amount of DNAs or proteins with different lengths or molecular weights. Your job is to separate them and figure out what they are. How will you do? Difficult or not?

Object : DNA Proteins

They are too tiny to be seen. The samples are too less to be handled.

Electrophoresis Technology
Powerful electrophoretic techniques have been developed to separate macromolecules on the basis of molecular weight. The mobility of a molecule in an electric field is inversely proportional to molecular friction which is the result of its molecular size and shape, and directly proportional to the voltage and the charge of the molecule.

Concept

Electrophoresis is used to identify and study charged molecules and macromolecules.

It is a method that separates macromoleculeseither nucleic acids or proteins- basis on the size, electric charge, and other physical properties.

Principle of Electrophoresis
The term electrophoresis describes the migration of charged particle under the

influence of an electric field. Depending on

the nature of the net charge, the charged particles will migrate to the electrode bearing opposite charge .

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Macromolecules are normally have charges

COO + H+ Pr + OHNH2

COO + H+ Pr + OHNH3+

COOH

Pr NH3+

PH>PI

PH=PI

PH<PI

(2)Forces which drive charged molecules moves

Electric force F=X(volt)Q
Resistense force

F=6

When they balanced:

F= F

QX=6

/X=Q/6

Factors affect the electrophoresis

U stand for /X called mobility
The rate of migration through the electric field depends on the strength of

the field, size and shape of the molecules,

relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving.

Factors affect the electrophoresis

1SamplesQsize , mass and shape.
Q V r V globin >fiber

2Strength of the field X V 3Buffer solutionpH determine Q(pH-PI)Q V

componentsstable
V .

(4) ionic strength 0.02-0.2MI current V

I current

Factors affect the electrophoresis

4Inert supporteasy to be stored no
electroosmosis 5Temperaturetoo highproduce heat and leads to vaporize water.

Note that big molecules travel slowly through the gel

matrix and small molecules travel more quickly, then they become separated according to their size and charge.

Equipments:

An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube.

After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other.

Classifications:
1.Cellulose acetate membrane electrophoresis 2. Polyacrylamide gel electrophoresis, PAGE 3. Agarose gel
Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study.

Content

Cellulose Acetate Membrane Electrophoresis of Serum Proteins

Cellulose acetate membrane electrophoresis

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Objectives:
1. Learn the basic principles of electrophoresis.

2. Learn how to use electrophoresis technology to

separate serum proteins.

Cellulose acetate membrane electrophoresis Principle

Cellulose acetate membrane serve as the inert

support. In barbital buffer pH=8.6 the PI of all

serum proteins is less than 8.6they are negatively

charged and move to the cathode.They are separated

on the basis of motility.

Total serum protein = albumin + globulin

Albumin is the protein of highest concentration in the serum. It carries many small molecules, but is also of prime importance in maintaining the oncotic pressure of the blood.
Globulins are roughly divided into alpha-1, alpha-2, beta, and gamma globulins. These can be separated and quantitated in the laboratory by electrophoresis and densitometry.

The alpha-1 fraction or portion includes alpha-1 anti-trypsin and thyroxine binding globulin. The alpha-2 fraction contains haptoglobin, ceruloplasmin, HDL, and alpha-2 macroglobulin.
The beta fraction includes transferrin, plasminogen, and betalipoproteins (see LDL). The gamma fraction includes the various types of antibodies (immunoglobulins M, G, and A). The gamma fraction includes the various types of antibodies (immunoglobulins M, G, and A).

pI and motility of serum proteins

Name

pI

motility

mass

Albumin
Globulin Globulin Globulin Globulin Globulin

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Procedures:
1. Load your sample on the membrane:
2. Place them to the box containing buffer: 3. Plug the electrode and turn on the power to run the samples: Adjust the volt(100v) and run for 1 hr. 4. Staining: 5. Washing:

Result

Analyze your result.

2 1

There are two basic types of materials used to make gels: agarose and polyacrylamide. The polyacrylamide gel electrophoresis (PAGE) technique was introduced by Raymond and Weintraub (1959). Polyacrylamide is the same material that is used for skin electrodes and in soft contact lenses.

Part II
Polyacrylamide gel electrophoresis
Of serum proteins

Polyacrylamide gel may be prepared so as to provide a wide variety of electrophoretic conditions. The pore size of the gel may be varied to produce different molecular seizing effects for separating proteins of different sizes. In this way, the percentage of polyacrylmide can be controlled in a given gel. By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2,000 kdal. This is the ideal range for gene sequencing, protein, polypeptide, and enzyme analysis.

Polyacrylamide gels can be cast in a single percentage or with varying gradients. Gradient gels provide continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. Because of this banding effect, detailed genetic and molecular analysis can be performed on gradient polyacrylamide gels. Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels.

Under the influence of an electrical field charged molecules and particles migrate in the direction of the electrode bearing the opposite charge. During this process, the substances are usually in aqueous solution. Because of their varying charges and masses, different molecules and particles of a mixture will migrate at different velocities and will thus be separated into single fractions.

Stacking gel

Separating, or resolving, gel

Regardless of the system, preparation requires casting two different layers of acrylamide between glass plates. The lower layer (separating, or resolving, gel) is responsible for actually separating polypeptides by size. The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel.

Procedures:
1. Polyacrylamide gel preperation (Two layers)
2. Lode the sample 3. Electrophoresis 4. Staining

Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals.

Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid.