particles and Peter Stein Nielsen for the plastocyanin clone. This protoirn. - + + te-. Tsr., work was supported by grants from The Norwegian Research.
\.1 1994 Oxford University Press
Nucleic Acids Research, 1994, Vol. 22, No. 15 3261 -3262
Enrichment of DNA-binding proteins from crude tissue for electrophoretic mobility shift assay using magnetic phospho cellulose particles Kristin Hollung, Odd S.Gabrielsen2 and Kjetill S.Jakobsenl,* Divisions of Botany and 'General Genetics, Department of Biology, University of Oslo, PO Box 1031 Blindern, N-0315 Oslo and 2Department of Biochemistry, University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway Received March 31, 1994; Revised and Accepted June 30, 1994 Analysis of DNA-binding proteins by electrophoretic mobility shift assays (EMSA) and footprinting has traditionally been performed with crude nuclear protein extracts. This approach involves isolation of nuclei, often dependent of Percoll gradient centrifugation, has the disadvantage of being time consuming and results in low yield of active extract. Thus, large amounts of starting material, typically 0.1 -1 kg plant tissue or two litres of mammalian cell culture are required (c.f. 1, 2, 3, 4). Studies of DNA-binding proteins from low-abundance tissues have therefore been difficult. Methods which avoid isolation of nuclei, for example by employing preparative chromatography of crude cell extracts, have been described, but these procedures are not less laborious compared to the conventional approaches (5). Recently, magnetic phospho cellulose particles have been developed which allow rapid protein purification and fractionation on a small scale (Ris0en and Gabrielsen, manuscript in preparation). We show here that magnetic phospho cellulose particles can be successfully used to isolate a protein fraction enriched in DNAbinding proteins, suitable for EMSA, directly from crude plant tissue. With this method we observe specific band-shifts, while utilizing 100-fold less starting material than previously reported for nuclear extracts. Protocol: Barley embryos are dissected from immature seeds (30 days after flowering) and 1 g is homogenized in a total volume of 10 ml extraction buffer (0.5 M NaCl, 50 mM Tris-HCI pH 8.0, 5 mM EDTA, 20% Glycerol, 20 mM 3-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF)). For leaves, 1 g is ground in a mortar with the same extraction buffer. The homogenate is forced through an Eton press at - 80°C to break the cells, and cell debris is removed by centrifugation at 40 K for 2 hours. Total protein extract is dialysed for 2 hours with dialysis buffer (0.1 M NaCl, 20 mM Tris-HCI pH 8.0, 1 mM EDTA, 10% Glycerol, 10 mM (3-mercaptoehanol, 1 mM PMSF) at 4°C with stirring, to lower the salt content. Typically, this protein extract will contain about 5-6 mg/ml protein for embryos and 1-2 mg/ml for leaf material (determined by the BioRad protein assay, BioRad) and can be stored at -800C. Twenty mg magnetized phospho cellulose (SCIGEN Ltd) is washed twice with 0.5 ml of a solution containing buffer A (20 mM Tris-HCI pH 8.0, 1 mM EDTA, 10% Glycerol, 1 mM DTT, 1 mM *
To whom correspondence should be addressed
PMSF) plus NaCl added to 0.1 M. After wash removal, extract containing 6-8 mg total protein is added and allowed to bind for 5 minutes on ice. A magnet suitable for microfuge tubes (MPC-E, Dynal A/S,) is used in all steps which involve the magnetic particles. After binding, the supernatant is discarded, and magnetic particles with bound protein are washed twice with buffer A + 0.1 M NaCl, before the positively charged proteins are eluted in 150 jil buffer A containing 1 M NaCl and 0.1% Triton X-100 by incubating 5 minutes on ice. Concentration of proteins with a Microcon-10 concentrator (Amicon) may be necessary to reduce the final NaCl content in the EMSAs. We have tested this approach for isolation of DNA-binding proteins from two different barley tissues, embryos and leaves, using different promoter fragments as probes in separate EMSAs. Figure 1A shows an EMSA with barley embryo proteins and a Lea B19. 1 promoter fragment (position -279 to -208) (6) containing a putative abscisic acid responsive element (ABRE). At least two complexes were seen on the gel, as was observed for the Em ABRE fragment, a B19 homolog in wheat, using nuclear extracts from wheat embryos (7). Using the same amount (2 jLg) of total extract no band-shift was observed. However, at longer exposures we could detect faint bands identical to those seen for the enriched extract. Nuclear extracts prepared according to (3) gave shifts of similar intensities as with phospho cellulose extract using the Lea promoter fragment. Figure lB shows an EMSA with barley leaf proteins and a promoter fragment (position -618 to -458) from the barley plastocyanin gene (8). In both experiments, binding to the probe sequences was abolished by competition with molar excess of unlabelled probe fragment, showing that the complexes were specific. Addition of similar amounts of non-specific DNA had no effect (not shown). Both promoter fragments shown here give the same shift as reported previously for the same experiments with nuclear extracts (7,8). However, we cannot rule out that differences in band-shifts with nuclear extracts and magnetic phospho cellulose extracts may be found for some other genes. Therefore, a comparison of the two approaches may be necessary when an 'uncharacterized' promoter is investigated by EMSA. Our results show that this procedure for purification of DNAbinding proteins is very effective for plant EMSAs using different
3262 Nucleic Acids Research, 1994, Vol. 22, No. 15 A
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Kent ME9 8AQ, UK for gift of magnetized phospho cellulose particles and Peter Stein Nielsen for the plastocyanin clone. This work was supported by grants from The Norwegian Research Council (NFR) to O.S.G. and K.S.J.
REFERENCES 1. Dignan,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Nucleic Acids Res.
11, 1475-1489. 2. Green,P.J., Kay,S.A. and Chua,N.-H. (1987) EMBO J. 9, 2543-2549. 3. Jensen,E.O., Mercker,K.A., Schell,J. and de Bruijn,F.J. (1988) EMBO J. 7, 1265-1271. 4. Echeverria,M., Delcasso-Tremousaygue,D. and Delseny,M. (1992) Plant J. 2, 211-219. 5. Segall,J., Matsui,T. and Roeder,R.G. (1980) J. Biol. Chem. 255, 11986-11991. 6. Stacy,R.A.P. and Jakobsen K.S. (1993) EMBL acc. no. X76933.
7. Guiltinan,M.J., Marcotte,W.R. and Quatrano,R.S. (1990) Science 250, 267 -271. 8. Nielsen,P.S. and Gausing,K. (1993) Eur. J. Biochem. 217, 97-104. 9. Gabrielsen,O.S., Hornes,E., Korsnes,L., Ruet,A. and Oyen,T.B. (1989) Nucleic Acids Res. 17, 6253 -6267. 10. Gabrielsen,O.S. and Huet,J. (1993) Methods Enzymol. 218, 508-525. 11. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Figure 1. A. EMSA with a promoter fragment from Lea B19.1 and barley embryo protein. B. EMSA with the RsaVI promoter fragment from the plastocyanin gene and leaf protein extract. Protein extract from embryo or leaves, purified with magnetic phospho cellulose, is included in the indicated lanes, and sequence specific competitor is added in molar excess as indicated. In Fig lA. the lane denoted Tot contains total protein extract (2 jig) prepared as described. The B19.1 71 bp promoter fragment was cloned in pBluescript and re-isolated as a SpeI/PstI fragment, plastocyanin 160 bp RsaVI fragment was in pBS and isolated as a EcoRI/XbaI fragment. Both fragments were end-labelled with a-32P-CTP and Klenow polymerase (Boehringer Mannheim) as described (11). Assay conditions are 2 lsg protein, 0.025-0.1 M NaCl, 10 mM Tris-HCl pH 8.0, 0.5 mm EDTA, 1 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF, 0.25-1 Ag poly [d(IC) *d(I-C)] as a non-specific competitor. Specific competitor was added in molar excess as indicated in the figure and was incubated with the assay mixture for 5 minutes at room temperature, before 20-30 fmol end-labelled probe was allowed to bind for 20 minutes. Separations are performed on a 4% polyacrylamide gel containing 0.025% glycerol in 0.5 xTBE electrophoresis buffer.
tissues and DNA probes. We have obtained 0.8-1 mg purified protein per gram barley embryos and 0.3-0.4 mg per gram leaves, enough to run 400-500 embryo EMSAs or 150-200 leaf EMSAs. In contrast, the yield for previously described methods varies from 1-13 EMSAs per gram starting material (2,3,4). The protocol described here has the advantages that it is not necessary to isolate nuclei, and the yield is 12 -500-fold higher compared to other methods, judged from the amount of starting material per lane. Therefore this approach can be used in situations where a limited amount of starting material is available, for instance cornparative studies of DNA/protein interactions between different cell or tissue types. This protocol may also be used as an initial fractionation preceeding the isolation of specific proteins with biotin end-labelled DNA- probes attached to magnetic beads (Dynabeads M-280 Streptavidin) as previously described (9,10).
ACKNOWLEDGEMENTS We thank Per Ame Ris0en for help with the magnetic protein purification, Solveig Hauge Engebretsen for technical assistance, Robin A.P.Stacy for comments on the manuscript, SCIGEN Ltd,
