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© 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 7 1815
A simple and efficient method for the purification of
specific DNA binding proteins
Susan E.Rutberg and Zeev Ronai*
Molecular Carcinogenesis Program, Naylor Dana Institute for Disease Prevention, I Dana Road,
Valhalla, NY 10595, USA
Submitted December 26, 1991
In an attempt to purify large amounts of a protein that
specifically binds to a newly identified UV-responsive element
(URE; 1) we have developed a simple method that enables
efficient coupling of specific target DNA sequences to a support
matrix. Although several procedures have been reported (2-4),
the efficiency of coupling the target DNA sequence to the support
matrix is routinely low or the procedures are highly specialized
and labor intensive to perform. In the newly developed procedure,
the DNA sequence of interest is initially tailed with poly(A)
nucleotides and subsequently coupled to oligo-dT cellulose. The
affinity columns prepared in this way are quick and simple to
construct and the attachment of the DNA to the column occurs
at the ends of the oligomer rendering the entire target sequence
available for protein binding.
In developing this procedure, a double stranded URE sequence
(TGACAACA) which has a two bp overhang at the 5' ends (30
fig) was tailed using 2 units of terminal transferase (Promega)
per ng of DNA in the presence of 0.2 mM dATP at 37°C for
1 hour. The enzyme was deactivated (65°C for 10'), and the
reaction mixture was passed through a G-25 (Pharmacia) column
prior to precipitation with ethanol. The DNA pellet was
resuspended in coupling buffer consisting of 10 mM Tris pH 8.0,
1 mM EDTA, and 1 M NaCl. 100 mg of oligo dT cellulose
(Boehringer Mannheim) was hydrated in the same buffer. The
resulting mixture was transferred to a Poly Prep Chromatography
Column (Bio Rad) and washed with 15 ml coupling buffer. The
URE-DNA was applied to the column with 6 - 8 passes of
solution through the matrix. Studies in which we have
incorporated 35S-labeled dATP during tailing of the DNA
indicate that the majority of coupling occurs during the first 3
passes and coupling efficiency of 98% are routinely achieved.
The column is subsequently washed with coupling buffer before
proceeding. Nuclear proteins were prepared from UV-treated rat
fibroblast cells through a procedure which leaves the proteins
in buffer consisting of 20 mM HEPES pH 7.9, 100 mM KC1,
0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT and 20% glycerol
(5). We have found it necessary to modify the composition of
this buffer by eliminating the DTT and reducing the glycerol to
less than 5% in order to optimize protein binding and minimize
loss of DNA from the affinity column. Subsequent to equilibration
with 15 ml of A50 buffer (50 mM Tris pH 8.0, 1 mM EDTA,
50 mM (NH^SC^ and 3% glycerol), 1-30 mg of nuclear
proteins are passed multiple times through the column for 3 hours
at room temperature or alternatively, overnight at 4°C using a
peristaltic pump set at low speed. The column is then washed
3 times with A-50 buffer (10 ml each) and the proteins are eluted
with 2 aliquots of 0.5 ml A-600 buffer (50 mM Tris pH 8.0,
1 mM EDTA, 600 mM (NH^SCv 10% glycerol and 0.5 mM
DTT). At this point, the column can be re-equilibrated with A-50
buffer and re-used multiple times or the DNA can be eluted from
the column with water and re-used for various other purposes.
Using this procedure we have obtained a yield of approximately
5 ng of purified 40 Kd protein from 20 mg of nuclear extract.
The profile of proteins before purification, retention of DNA on
the oligo dT column and activity of the purified proteins in
electrophoretic mobility shift assay (EMSA) are shown in Figure
1.
ACKNOWLEDGEMENTS
We wish to thank Yang Ming Yang and David Wivagg for their
contributions of nuclear proteins in this study. This research was
supported by grants from the A.C.S., N.C.I, and N.I.E.H.S.
REFERENCES
Levens.D. and Howley.P.M. (1985) Mol. Cell. Biol. 5, 2307-2315.
Arcangioli.B. el al. (1989) Eur. J. Biochem. 179, 359-363.
Kawaguchi.H. el al. (1989) Nucleic Acids Res. 17, 6229-6240.
Ronai.Z. and Weinstein.I.B. (1990) Cancer Res. 50, 5374-5381.
DignamJ.D. et al. (1983) Nucleic Acids Res. 11, 1475-1489.
i'
Figure 1. (I) Profile of proteins prior to (A) and after (B) purification separated
by 10% SDS-polyacrylamide gel electrophoresis and stained with Coomassie
blue, (II) retention of DNA on the oligo dT affinity column as determined through
monitoring the amount of •>3S-labeled taikd DNA thai was washed off the column
at the indicated stages and (111) activity of proteins purified through this procedure
as determined through EMSA using *2P-labeled URE target DNA reacted with
approximately 100, 200, 400 and 800 pg (lanes 1 - 4 ) of purified protein yielding
approximately 75-fold increase in binding activity over that achieved with 7.2
and 14.4 jig of nuclear extract (shown in lanes 5 and 6, respectively) as determined
through quantitation using a radio-imaging blot analyzer (AMBIS). Shown in lane
8 is the migration of probe alone.