Biology Reference
In-Depth Information
proteins and nuclear proteins are of particular pharmacological
interest, a suitable extraction procedure is desirable that selectively
harvests these proteins (which are frequently present in very low
quantities) into distinct subcellular fractions, separated from high-
abundance proteins present in cytosol or cytoskeleton. The method
of sequential extraction of cell content is based on the different
solubility of certain subcellular compartments in special reagent
mixtures (
6
). Sequential extraction results in a stepwise disintegra-
tion of the cell's structure, yielding four subcellular fractions (
6
).
Upon treatment with the fi rst extraction buffer, cells shrink as a
result of the release of the cytosolic content but remain intact in
their overall structure. After the second extraction step, due to the
solubilization of membranes and membrane organelles, only iso-
lated nuclei and the cytoskeleton remain intact. The treatment of
the residual material with the third extraction buffer destroys the
structure of the nucleus, thus probably solubilizing the nuclear
proteins. Finally, the cytoskeleton components are liberated during
the fourth and fi nal extraction.
In the application of difference gel electrophoresis (DIGE),
different samples are labeled with fl uorescent dyes of similar mass
and identical charge and are then separated on the same 2D gel.
The standard sample included in each gel comprises equal amounts
of each sample to be compared and was found to improve the
accuracy of protein quantifi cation between samples from different
gels, allowing accurate detection of small differences in protein
levels between samples.
Image analysis is automatically performed with minimum user
intervention with DeCyder Differential Analysis Software which
comprises the four modules listed below.
DIA (differential in-gel analysis): protein spot detection and quan-
tifi cation on a set of images, from the same gel. Features include
background subtraction, in-gel normalization, and gel artifact
removal.
BVA (biological variation analysis): matching of multiple images
from different gels to provide statistical data on differential
protein abundance levels between multiple groups.
Batch Processor: fully automated image detection and matching of
multiple gels without user intervention.
XML toolbox: extraction of user-specifi c data facilitating automatic
report generation (
7, 8
).
The DIA module quantifi es the spot volume for each image and
expresses these values as a ratio, thereby indicating changes in abun-
dance by direct comparison of corresponding spots. This ratio
parameter can be used, in a small-scale experiment, to directly evalu-
ate changes between two labeled protein samples (e.g., control and
drug-treated samples). Alternatively, the ratio can be used for protein
Search WWH ::
Custom Search