We have developed a simple and rapid method for preparing DNA-binding protein extracts from mammalian cells. The protocol is derived from the large scale procedure of Dignam et al.(1) and utilizes hypotonic lysis followed by high salt extraction of nuclei. The technique described by Dignam has several drawbacks, including the need for large numbers of cells and lengthy incubation and dialysis steps. It is labor-intensive and precludes preparation of multiple samples simultaneously. Our aim in developing this micropreparation procedure was to easily and rapidly extract DNA-binding proteins from small numbers of cells. Frequently, the quantity of cells available for extraction of DNA-binding proteins is limiting, as in analysis of clinical samples, of multiple clones of transfected cells, or of COS cell pools transiently transfected with a cDNA expression library. Ideally, such a technique would allow processing of many samples simultaneously and quickly on thebenchtop. The method described in this report accomplishes these goals. In addition, it gives an excellent yield of DNA-binding proteins, comparable to that ofthe large scale Dignam protocol with minimal proteolysis.

We typically start with between 5x105 and 107 cells. All centrifugations of less than 30 seconds are carried out in a room temperature microfuge; between steps, the samples are placed on ice. Adherent cells are scraped into 1.5 ml of cold phosphatebuffered saline (PBS); non-adherent cells are pelleted and resuspended in 1.5 ml cold PBS. The cell suspension is then transferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 Alcold Buffer A (10 mM HEPES-KOH pH 7.9 at 4 C, 1.5 mm MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube. The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 20-100/d (according to starting number of cells) of cold Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 minutes at 4 C and the supernatant fraction (containing DNA binding proteins) isstored at-70 C. The yield is 50 to 75 ltg protein per 106 cells.