But we noticed that a minor reduction of the divalent cation concentration led to a reversible separation of the chromatin strands by charge repulsion. It was of importance to demonstrate loops at the chromatin level in unextracted chromosomes. We developed several techniques for this purpose. Human chromosomes were spread at low ionic strength resulting in the unfolding of the basic chromatin fiber to its more extended form. The surface structure of a chromosome observed with the scanning microscope is also consistent with chromatin loops.
Loops emanate from a central region where they appear tethered by an organizer, the scaffolding.
Note that not all loops can be tracted completely, due to contaminating material adhering to the chromosomal periphery. Also, despite a predominatly radial orientation of the loops, certain fibers run out of the plane of cross-sectioning. Indeed, although necessitating a number of years of research, we were able to successful answer both questions. The difficulty of finding specific scaffold proteins was formidable, but we made an important observation that allowed us to identify potential scaffolding proteins biochemically with some confidence. Based on the assumption that scaffold proteins were solubilized from soft but not from robust chromosomes during the extraction procedure, we could identify two candidate scaffold proteins by comparative biochemistry of the two different chromosome preparations.