Biochemical instrumental analysis - 8 Dr. Maha Al-Sedik 2015 CLS 332

Procedure 1- Medium Saturation 2- Sample application 3- Running the sample 4- 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 electrophoresis Zone 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 electrophoresis vertical on the first one

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