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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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