advances-in-protein-chemistry

protein monitoring during disease development and screening of potential drugs for their effect on the expression of protein. However,
the expertise and cost involved has so far limited its use as a routine technique. The overall workflow in 3D electrophoresis is summarized
in Figure 10.
Figure 10: Workflow of 3D gel electrophoresis system. [Adapted from http://www.3d-gel.com].
Ultracentrifugation
Ultracentrifugation is referred to as centrifugation at very high speeds which may go as high as 2,000,000xg. Due to the enormous
centrifugal force applied, it is possible to separate proteins and other macromolecules by this technique.
Density gradient centrifugation involves a linear concentration gradient of sugar (sucrose), glycerol, or commercially available silica
based density gradient media like Percoll (a trademark owned by GE Healthcare) is generated in a tube such that the highest concentration
is on the bottom and lowest on top. The concentration gradient also behaves as density gradient as the density also increases with
concentration. The sample is layered on top and centrifuged at ultra-high speeds. The heavier molecules migrate towards the bottom
faster than lighter ones. When the proteins move through a density gradient, they encounter liquid of increasing density and viscosity,
which counteract the increasing centrifugal force and the proteins are segregated into sharp bands or zones. If the centrifugation is
performed in the absence of sucrose, as particles move farther and farther from the center of rotation, without getting concentrated in
sharp bands. Once the centrifugation is over, the gradient is then fractionated and different bands are collected.
The disadvantages in ultracentrifugation are cross-contaminations among fractions. This may sometimes result in relatively poor
yields and low resolution. In addition, Takeuchi and Saheki [20] have reported the problem of removal of lipids and apoproteins during
ultracentrifugation of lipoprotein particles, though it may not be considered as a disadvantage in general terms.
Acknowledgment
The author wishes to thank Dr. Fahim H. Khan and Dr. Haseeb Ahsan, for their support.
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