Electron Microscope Visualization of Chromatin and Other Dna-Protein Complexes

Abstract

In this review we have tried to cover the direct mounting techniques currently in use and, through micrographs kindly supplied by our colleagues, to illustrate uses of the different methods. For the novice, it is important to select one technique, taking time to become acquainted with its use in visualizing simple test samples before applying it to examine more complex structures. We have tried to emphasize the importance of controlling the fixation for two reasons. First, confidence in the fixation step allows one to correlate structures observed by EM with structures present in solution. Second, fixation of otherwise labile samples allows one to apply powerful purification techniques.

Human Claspin Is a Ring-shaped DNA-binding Protein with High Affinity to Branched DNA Structures

“…5, A and B). We incubated these substrates with Claspin, and then the samples were prepared for electron microscopy (EM) as described elsewhere (16).…”

“…The samples were fixed by the addition of glutaraldehyde to a final concentration of 0.6% for 10 min at 25°C and chromatographed on 1-ml Bio-Gel A50m columns to remove unbound proteins. Fractions containing DNA-protein complexes were prepared for electron microscopy as described previously (16). The samples were examined on a Phillips CM12 instrument, and images were scanned using Nikon-4500 film.…”

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

“…We purified primate cell-replicated DNAs as described in the legend of Fig. 5 and mounted them for electron microscopy as described 38,50 . Briefly, the indicated gel-purified DNAs were mixed in a buffer containing 2 mM spermidine, adsorbed to glow-charged carbon-coated grids, washed with a graded water/ethanol series and rotary shadow cast with tungsten.…”

DNA looping by Ku and the DNA-dependent protein kinase

“…After chemical fixation with 0.6% glutaraldehyde, reaction mixtures were chromatographed on a 2-ml bed volume of Bio-Gel A-15m (Bio-Rad). Column fractions were mounted by the spermidine mounting technique, dehydrated in a graded series of ethanol solutions, and rotary shadow cast with tungsten (50,51). Samples were imaged and photographed on a Philips CM30 at an accelerating voltage of 50 kV.…”