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A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis
OSSIPOW, Vincent, LAEMMLI, Ulrich Karl, SCHIBLER, Ulrich
OSSIPOW, Vincent, LAEMMLI, Ulrich Karl, SCHIBLER, Ulrich. A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis. Nucleic Acids Research , 1993, vol. 21, no. 25, p. 6040-6041
DOI : 10.1093/nar/21.25.6040
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6040-6041 Nucleic Acids Research, 1993, Vol. 21, No. 25 © 1993 Oxford University Press
A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis
Vincent Ossipow, Ulrich K.Laemmli and Ueli Schibler*
Departement de Biologie Moleculaire, Sciences II, Universite de Geneve, 30, Quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland
Received October 13, 1993; Accepted October 17, 1993 SDS-polyacrylamide gel electrophoresis has proven to be the method of choice for the separation of proteins according to their size (1). This method has been applied to the size estimation of transcription factors present in crude nuclear extracts.
Transcription factors can be detected among gel-fractionated proteins by virtue of their ability to bind to specific DNA sequences. For this purpose, mainly three protocols have been employed: 1. UV-crosslinking of radiolabeled DNA recognition sequences to the proteins prior to electrophoresis (2); 2.
'Southwestern blotting', in which gel fractionated proteins are transferred from the gel to a membrane and, after renaturation in situ, are probed with a specific radio-labeled DNA recognition sequence (3); and 3. elution of size separated proteins from polyacrylamide slices followed by their analysis by electrophoretic mobility shift assays (EMSA's) or other convenient techniques (2, 4). While the simplicity and rapidity of the first two methods render them attractive, they are accompanied by several drawbacks. The efficiency of UV-crosslinking varies greatly between different protein:DNA complexes, depending on the chemical nature of amino acid-base interactions established between the protein and DNA partners (5). Moreover, this method shifts the protein-DNA binding equilibrium towards complex formation, particularly if long crosslinking times are required. Thus, it does not necessarily allow for affinity measurements and may reveal weak unspecific interactions, if crude extracts are used. A serious shortcoming of Southwestern blot analysis is that it is limited to the analysis of proteins which bind as monomers or homopolymers, since the subunits of obligatory heteropolymers are separated during SDS-gel electrophoresis. Moreover, as in the case of covalent crosslinking, affinity measurements are not readily feasible using this technique.
Elution and renaturation of SDS-gel fractionated proteins can overcome the problems encountered with UV-crosslinking and Southwestern blotting experiments, but are labor-intensive.
According to published procedures (6), proteins are eluted from the gel pieces with SDS—buffer, precipitated with acetone, denatured with concentrated guanidium hydrochloride (Gd-HCl) solutions and renatured by the removal of Gd-HCl by gel filtration, dialysis or dilution. Unfortunately, these lengthy de- and renaturation procedures often result in low recoveries of active DNA-binding proteins and may require a prepurification step to ensure a sufficient amount of starting material. Moreover,
while practicable for a small number of samples, this method becomes cumbersome if large numbers of gel slices have to be handled.
Here we describe a very simple and efficient method to recover active DNA-binding proteins from SDS-gels. This single step elution-renaturation procedure is based on the observation that mild non-ionic detergents, such as Triton X-100, remove SDS from protein—SDS complexes and sequester it into micelles that no longer interfere with DNA binding.
The methodology is as follows: nuclear extracts are incubated in sample buffer (1) for 10 min. at 37°C, and fractionated by gel electrophoresis (1). As judged from Coommassie blue stained gels, this mild SDS treatment results in an identical protein pattern as boiling. The gel strips containing the fractionated proteins are cut transversely with a razor blade into the desired number of fractions. Each gel piece is then placed into a 1.5 ml Eppendorf tube containing three volumes (per weight of the polyacrylamide slice) of elution—renaturation buffer and homogenized with a small Teflon or stainless steel pestle. After a four hour incubation period at 37 °C, the residual polyacrylamide is sedimented in an Eppendorf microfuge, and the supernatant (about 60% of total volume) is transferred to a new tube. This eluate can be used directly in electromobility shift binding assays.
In Figure 1 we show the application of the technique to a search for dimerization partners of DBP, a bZip transcription factor (for review see 7), among size fractionated liver nuclear proteins.
Aliquots of the supernantants of each fraction were incubated at 37 °C with an excess of the DBP bZip domain (amino acids 211 to 325 overexpressed in E.coli, see Figure legend). The molar excess of the DBP bZip polypeptide should drive any protein with a DBP-compatible dimerization domain into heterodimers.
Such heterodimers can then be visualized as protein/DNA complexes that migrate with a lower mobility in EMSA's as compared to DBP bZip homodimers. As shown in Figure 1A, multiple fractions contain protein:DNA complexes in addition to the DBP bZip homodimers. That these complexes indeed reflect heterodimers with DBP Zip is indicated by gel shift experiments, in which die relevant size fractionated proteins are incubated with the binding site in the presence or absence of excess DBP bZip (Figure IB).
The same protocol is also applicable to proteins with DNA recognition domains other than bZip. Figure 2 shows an experiment, in which a size fractionated crude liver nuclear
* To whom correspondence should be addressed
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Molecular
Might IltD*] W 45 34 V) 24
I l i l i
fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 U 17 18 1? 20 21 22 23 2k homo and heterodimers
— bZIPDBP homodimers
Nucleic Acids Research, 1993, Vol. 21, No. 25 6041
Molecular
weight [kDa] 44 45 34 traction 1 2 3 4 5
24 20 14 i I I 7 8 9 10 11
Prolein/DNA complexes
— - free DNA
B
fraction 7 7 10 10 11 11 12 12 U H 16 l i 17 17 20 20 bZIPDBP . - , . , . < • - • • - * - • - * .
homo and heterodimers
— bZIP DBP homodimers
Figure 2. Detection of octamer binding proteins in a gel size-fractionated rat liver nuclear extract. 15 /ig of a liver nuclear extract were fractionated on an 12% SDS-polacryamide mini gel (gel slot: Width: 5 mm:, length 55 mm, thickness: 1.5 mm). The gel cut into 11 fractions that were treated as described above (Figure 1A), and 10 pi aliquots were examined for the presence of octamer binding proteins by EMSAs (6.5% polyacrylamide gel). The binding reaction (final volume 20 /J) contained 8 ng of a double-stranded oligonucleotide spanning the octamer site of the immunoglobulin heavy chain promoter (12) and 1 ng of poly dldC.
This research was supported by the Swiss National Science Foundation and by the State of Geneva.
Figure 1. A. Search for dimerization partners of DBP in a gel fractionated rat liver nuclear extract. 30 pg of crude rat liver nuclear proteins prepared as described (9) were heated for 10 min at 37 °C in SDS-PAGE loading buffer, and separated in a SDS 11 % polyacrylamide slab gel. The gel slot (width: 7 mm:, length 120 mm, thickness: 1.5 mm) was divided into 24 fractions of 40 mg. Gel pieces were homogenized in 3 volumes (120 id) of ERB (elution-renaturation buffer: 1%
Triton X-100, 20 mM Hepes pH 7.6, 1 mM EDTA, 100 raM NaCl, 5 mg/ml BSA, 2 mM DTT, 0.1 mM PMSF, 0.1% Trasylol) and incubated at 37°C for 3 hours. The polyacrylamide residues were sedimented by centrifugation in a microfuge for 10 min. 10 pi of each supernatant was increased to 15 pi by the addition of 2 pi of (200 mM Hepes pH 7.6, 50 mM MgCl2, 340 mM KC1) and 3 pi of a 400 nM solution of the DBP bZip region (amino acids 211 to 325 inserted into the T7-expression vector pET-3b and overexpressed in BL21 pLysS, as described in 10). After an additional 15 min incubation at 37°C 5 pi of 10 mM Tris-Cl pH 7.5, 10 mM NaCl, 1 mM EDTA containing 2.5 ng of a double- stranded -"P-labeled oligonucleotide spanning the fp2 site (9) and 500 ng of poly dldC were added. Incubation was continued for 5 min at 37°C and 15 min on ice before DNA/protein complexes were analyzed by EMSA on a 7.5% non denaturating polyacrylamide gel as described (11). The molecular weights indicated correspond to molecular weight size markers run alongside the nuclear extract in the SDS 11 % polyacrylamide gel. B. Homo- and heterodimerization of DBP partners. 10 pi of each indicated fraction was incubated either with (+) or without (—) bZip DBP and the resulting protein/DNA complexes were examined by EMSA (6.5% polyacrylamide gel).
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extract was examined for proteins binding to an octamer recognition sequence. This analysis clearly reveals four proteins of different molecular masses that specifically bind to this DNA sequence element. Most likely, the largest protein corresponds to the ubiquitous transcription factor Oct-1 (8).
Given the speediness, simplicity, and reproducibility of this procedure, it should considerably facilitate the analysis of protein:DNA and protein:protein interactions, in particular when purified proteins are not available.
ACKNOWLEDGEMENTS
We would like to thank Walter Schaffner for the octamer oligonucleotide, Eileen Falvey for critical reading of the manuscript, and Nicolas Roggli for preparing the illustrations.
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