Biomedical Engineering Reference
In-Depth Information
10.1.4.2 Gel Electrophoresis
To achieve a separation, it is then necessary to use a sieving matrix that slows down
the largest molecules. Most commonly this is a gel made from polyacrylamide or
agarose whose mesh size (from a few tens of nanometers to a few hundred nanome-
ters) conditions the separation.
Practically, the solution containing the mixture to be analyzed is deposited at
one extremity of a gel slab. The whole slab is immersed in the buffer and an electric
field (up to ~ 100 V/cm) is applied via electrodes immersed in it. In these experi-
ments, the electrodes are physically separated from the sample (although in electri-
cal contact with it via the buffer solution) thus the electrochemistry at their surface
(electrolysis in particular) has no consequence. After applying the electric field for a
certain time, the molecules are stained and appear as bands (Figure 10.4).
The migration of small molecules within the gel can be modeled by comput-
ing the free volume available to the particle [9-11]. In this approximation, the size
R
g
of the chain is smaller than the distance between the crosslinks of the gel and
the DNA chain remains basically undeformed during the electrophoresis (Ogston
regime). This volume depends on the gel characteristics as well as on the particle
effective volume and one gets ultimately the following expression for the mobility:
(10.18)
µ
µ
1/ log
N
This equation is indeed well verified, and routinely used, for relatively short
molecules.
However, longer molecules deform in the gel and take the average orientation
of the electric field. They can be modeled by reptating chains within an array of
obstacles and their mobility becomes independent of the molecular weight again
(Figure 10.5) [11-13].
Figure 10.4
Electrophoregram of a “DNA ladder” showing the logarithmic dependence of the
mobility on the molecular weight (eq. 10.18).
