Quantitative Analysis of Chromatin Higher-Order Organization Using Agarose Gel Electrophoresis
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1038
In the past five years, there have been numerous molecular and genetic investigations of the mechanisms by which chromatin condenses into interphase chromosomal fibers (
1
,
2
). In addition to traditional molecular techniques such as electron microscopy (
3
–
5
) and analytical ultracentrifugation (
see
Chapter 9 ;
ref.
6
), it has recently been demonstrated that agarose gel electrophoresis can be used to quantitatively analyze the conformational and configurational changes of chromatin (
7
–
9
). The electrophoretic method requires neither large amounts of sample nor a large investment in equipment, making this structural technique potentially more accessible to the typical chromatin researcher. The key to “quantitative” agarose gel electrophoresis is to perform the experiment in a multigel (Fig. 1 ;
see
Subheading 1.3. ). The multigel approach was developed by Serwer (
10
) and previously has been used to characterize spherical and rod-shaped bacteriophages (
11
–
12
). It yields a measure of three important structural parameters: shape, flexibility, and surface charge density. When applied to chromatin, these parameters provide sensitive assays for nucleosomal subunit density, the spacing regularity of the nucleosomal array, and the average extent of chromatin folding (
6
,
7
–
9
). Together, this information can provide unique structural insight into chromatin higher-order organization.
Fig. 1.
(
A
) Schematic illustration of a 9-lane multigel. The view is from above after completion of each of the steps described in
Subheadings 3.1.
and
3.2.
Increasing agarose concentrations of the different running gels are represented by increased shading. The position of the chromatin bands relative to the bacteriophage T3 bands are illustrated for a 12-mer nucleosomal array. (
B
) Schematic diagram of a 9-lane slotformer viewed from the side.
