Biochemical instrumental analysis - 8

Dr. Maha Al-Sedik2015

CLS 332

Electrophoresis-2

Procedure1-

Medium Saturation

2-

Sample application

3-

Running the sample4-

Removal of supporting media

5-

Staining of compound

6-

Quantitation of protein zones

Medium Saturation

If the supporting medium is not gel , it must be saturated with buffer before electrophoresis start, So it can connect the current.

Sample application

The sample is applied by micropipette.

If the components of a mixture have opposite charge and they

are expected to move toward both electrodes , the application

will be central .

If the components of a mixture have common charge , The

application will be to the same side of charge.

Running the sample

The power is switched on at the specific voltage for the suitable time.

Removal of supporting media

Paper, cellulose acetate strips, gel and thin layer plates can be removed and air dried.

Staining of compound

Most biological compounds are not colored, it is necessary to

visualize them in order to determine their position on the

supporting medium after separation.

It is done by staining the media with a dye which will selectively

stain the components in the medium.

The amount of dye will be related to the amount of protein

present in the zone.

Most common used stains for protein are

nigrosine in acetic acid, amido black or

promophenol blue.

Most common for lipoprotein staining is sudan

black.

Quantitation of protein zones

There are 2 ways for quantitation:

1- Densiometry:

The supporting medium is fixed and cleared then

moved across a light beam.

The densiometer measures the amount of light

transmitted.

The light transmitted will be inversely proportional to

the concentration of protein.

GS-900 Calibrated Densitometry

2- Elution then spectrophotometric measurement:

Elution of dye from individual zone and subsequent

spectrophotometric measurement of eluted dye.

The elution method involves cutting the relevant area

of supporting medium into separate zones and elution

of the dye in each zone.

A- paper: its use now is limited because it takes a lot of time.

B-Thin layers: of silica, alumina or cellulose can be prepared on glass

plate.

C- Cellulose acetate: The membrane we buy are dry , opaque and

brittle that cracks easily if not handled gently. When the film is

placed in buffer the air spaces is filled with fluid and the film

becomes quite pliable.

Its great advantage is the speed of separation , and stability of the

results.

D- Gels: Agar, starch must be prepared in buffer shortly before use.

Support media:

The media may affect the separation in some manner :

1- Adsorption: Retention of sample molecule in the supporting media. It

increases with paper but disappear with cellulose acetate.

It is maximum with paper , and minimum with cellulose

acetate.

Surface of gel is negatively charged when contact with water because of the adsorption of hydroxyl ions.

Surface gel ions are immobile. Positive buffer ions orient with negative surface ions =

positive ionic cloud. Ionic cloud is mobile. Electrical current causes positive ionic cloud to move toward

the cathode.

2- Electroendosmosis:

Molecules that hold a weak negative charge or small size will not move.

Macromolecules (proteins) that have a sufficiently strong enough charge are able to oppose the flow of the positive ion cloud and move in the opposite direction towards the electrode of opposite polarity.

It is minimal in starch gel or polyacrylamide gel.

Types of electrophoresisZone electrophoresis

High voltage electrophoresis

Gel electrophoresis

Two dimensional electrophoresis

Zone electrophoresis

Any electrophoretic technique in which components are separated

into zones or bands in a buffer, and stabilized in solid, porous, or

any other support medium eg, filter paper, agar gel, or

polyacrylamide gel.

High voltage electrophoresis:

When low molecular weight proteins are separated by low

voltage paper electrophoresis, considerable diffusion occur, so

we use high voltage electrophoresis.

It produces so much heat , so direct cooling system is required.

Do not forget cooling system

Gel electrophoresis:

Gel electrophoresis uses a gel as a medium during

electrophoresis.

Gels suppress the thermal convection caused by application of the

electric field.

Gels can also simply serve to maintain the finished separation, so

that a post electrophoresis stain can be applied.

Gel electrophoresis

Types of gel:I Agarose: Cheap , non toxic .

Suitable for staining after separation.II Polyacrylamide: Prepared immediately before use from number of highly toxic

synthetic materials.

Pore size is controlled by modulating the concentrations of

acrylamide.

III Starch: Prepared by heating and cooling a mixture of partially hydrolyzed

starch in appropriate buffer

The starch is slightly more opaque than acrylamide or agarose.

Typical starch gel concentrations are 5% to 10%.

Effect of agarose concentration on migration of the DNA

Two-dimensional electrophoresis

First electrophoresis

Cut the zone of gel

Second electrophoresisvertical on the first one

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