Common Support Media for Biological Molecules

8/8/2019 Common Support Media for Biological Molecules

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Common support media forbiological molecules

PROTEINS.Native gel (acrylamide or starch)

Denaturating (SDS) gel (acrylamide).

Nuclic acids.Agarose gel

Acrylamide gel.


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Making a separation

Electrophoresis systems are designed to optimize the separation of specific

molecule types based on specific molecular parameters:

Nucleic acids: charge/BP is a constant. Separation can be based on numberof base pairs (given all molecules have the same shape). Larger molecule move

slower due to friction with gel.

Proteins: charge varies as a function of amino acid composition and buffer pH.Separation is based on charge/MW (shape may also vary) the exact combination of

factors varies for each molecule.


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Separating proteins based onMW

the problem associated with protein separation that too many separation parameters

involved has been solved by using denaturating SDS gels.

The proteins are heat denaturated which makes them all the same shape (linear).

The proteins are coated with an ionic detergent (SDS) which gives all molecules

approximately the same overall negative charge.

Separation is based on MW alone.


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Separation of DNA molecules in

agarose gels

In most cases the molecules are linear.

The phosphate groups bear negative charge at neutral pH (2 phosphates/BP)

Therefore the mobility will be based on number of base pairs/molecule.


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Principles ofSeparation

Before CE analysis , the analyst chooses an appropriate buffer to give a pH where

the solute is charged. For example, acidic compounds dissociate at high pH and

produce negatively charged anions. Conversely, basic compounds become

protonated at low pH and become positively charged cations.

The analyst injects a small volume of sample into the end of the capillary furthest

away from the detector. The capillary is then dipped into buffer-filled vials and avoltage applied. The ions move at a different speeds towards the electrodes, and

separation occurs depending on the number of charges and the size of the solute.


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The components

Power supply.this is usually a reversible polarity unit capable of producing up to 30 kV.

Sample introduction.there are two modes of injection:

Hydrodynamic mode:

in this method, the capillary is placed in the sample solution which is then raised

to a given height for a set time, whereby the sample flows into the capillary. Most

commercial instruments use either a vacuum or pressure as a means of introducing

the sample into a capillary, and this is much more reproducible. In the case of

capable of introducing precisely reproducible nanolitre volumes of sample into

the capillary.

Electro kinetic injection:

here, the analytical capillary is placed in the sample solution and a voltageapplied to cause the sample to flow into the capillary. Following the sampleintroduction, the capillary inlet is returned to the running buffer and electrophoresisis commenced.


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Capillaries.Capillaries generally consist of fused -silica protected by an external coating of polyimide.

The most common internal diameters are in the range of 50-100m. In order to perform on-line solute detection, a small window is burnt at the appropriate position , usually bycautiously placing the capillary in a small falme or heating element.

Oven .temperature control is very important in CE in order to achieve reproducible

results.

detector,.Although there are other detectors available for CE as in HPLC , by far the most

common is the variable wavelength UV detector. However, because the cell path

length is in effect the diameter of the capillary, which is typically only 50-100m,this method is often lacking sensitivity. Because the path length is so small, the lack

of sensitivity can often be solve by using a Z-cell that effectively increases the

optical path length many times. Although the sensitivity can be increased by 10-15

times, some efficiency is lost as a result of dispersion of the light.


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Forces acting on ions in solution

under the influence of an electricfield

Fe= electromotive force acting on ion.

Fd=frictional drag in medium (viscosity of electrolyte)

Fret=retardation effect caused by attraction of counter ions to opposite electrode.

Frel=relaxation effect (charge density in front > behind: electrostatic forces

attempt to rebuild/redistribute so (Frel) is formed (balancing)


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Electrophoretic mobility

V=e.E 1

Fd=6rv 2

Fe=qE 3

At steady state Fd=Fe

6rv=qE 4

Solving for V and substituting into 1

e=q/ 6rv

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Common Support Media for Biological Molecules