PNL Volume 17 1985
R. Henschke and E. Nucken Institute of Genetics
University of Bonn, Federal Republic of Germany
Until recently the most useful method of studying DNA binding pro-
teins employed radioactive labelled DNA (1, 2, 3). Working on DNA
binding proteins in chloroplasts of Pisum sativum, we have developed a
simple method which operates without labelled substances. This test is
suitable for detecting the DNA binding ability of diverse protein frac-
tions during the isolation procedure.
Assay procedure. 100 mkl of the DNA-free protein sample (in TEDP-
buffer containing 20 mM Tris/HCl pH 7-4, 1 mM ethylenediaminetetraacetic
acid, 1 mM dithiothreitol, 0.5 mM phenylmethansulfonyl fluoride, 0.05 M
NaCl) are incubated with 20 mkl of double stranded calf-thymus DNA (2 mg
DNA/ml H20) for 10 minutes at 30C. Then 50 mk1 of the mixture are suc-
tioned through a nitrocellulose filter (Schleicher & Schull, BA 85, 0.45 m,
pretreated by washing with 3 ml H2O). In this step proteins bind to
the nitrocellulose, whereas free double stranded DNA molecules pass
through the filter. If samples contain binding proteins, DNA-protein
complexes are formed and so nucleic acid molecules will be retained on
the nitrocellulose. To remove unspecifically bound DNA, the filter is
washed two times with 3 ml of TEDP-buffer. The filter is dried at 50C
for 10 minutes and incubated in ethidium bromide solution (10 mkg 2,7-di-
amino-10-ethyl-9-phenyl-phenanthridiumbromide/ml) for two minutes. Ethi-
dium bromide intercalates into the DNA, which can be detected by UV-light
induced fluorescence. Under these conditions only the spots of those
fractions containing DNA binding proteins are visible.
In addition, the following standards are necessary:
a) Positive control. 100 mkl of purified DNA binding proteins
(calf histones, 250 Pg/ml, a kind gift from M. Herlt) are incubated with
20 1 of DNA solution (2 mg/ml). 50 pi of this mixture are assayed.
b) Negative control. Corresponding to the positive control, lOOmkl
of a non-DNA binding protein (bovine serum albumine, 50 mg/ml) are incu-
bated with DNA.
Furthermore, the sensitivity of the assay was determined by dif-
ferent concentrations of histones. Even 10 mkg DNA binding proteins per
100 mkl were detectable.
The described test is useful to discover DNA binding proteins both
from the nucleus and from the organelles.
1. Tsai, R. L. and H. Green. 1973. J. Molecular Biol. 73:307-316.
2. Kohiyama, M., R. Kollek, W. Goebel, and A. Kcpes. 1977. J.
Bacterial, 129(2):658-677.
3. Jacq, A. and M. Kohiyama. 1980. European J. Biochem. 105:25-32.