Immunodepletion EMSA: a novel method to identify proteins in a protein-DNA complex

1995 Oxford University Press

Nucleic Acids Research, 1995, Vol. 23, No. 16 3345-3346

Immunodepletion EMSA: a novel method to identify proteins in a protein-DNA complex Roy B. Dyer1 and Norbert K. Herzogl92,3,* Departments of 1Microbiology and Immunology, 2Pathology and the 3Center for Tropical Diseases, The University of Texas Medical Branch, Galveston, TX 77555-0605, USA Received June 27, 1995; Accepted July 19, 1995

The electrophoretic mobility shift assay (EMSA) is a sensitive and powerful method to study the DNA binding activities of selective transcription factors in vitro (reviewed in 1). The discovery of transcription factor families and cross-dimerization of distinct family members stimulated the design of methods to identify the protein components of a DNA-protein adduct. Several procedures utilizing antibodies can identify proteins in DNA-protein complexes: (i) immunoprecipitation of UV crosslinked DNA-protein adducts; (ii) immunoblotting of an EMSA gel (2,3); (iii) band retardation by antibodies added to the protein-DNA binding reaction (supershift assay) (4). While these procedures localize a DNA binding protein to a particular complex and may imply the existence of a dimer, they do not actually prove the existence of a protein-protein interaction. We have designed a method that combines immunodepletion with the EMSA/supershift assay (IDEMSA) to analyze protein dimers of the NF-dKB/Rel family during LPS-induced differentiation of the 70Z/3 pre-B-cell line. The NF-KB/Rel transcription factor family modulates B-cell differentiation as well as a multitude of immune and acute phase response genes in a variety of cell types (reviewed in 5). NF-KB is the prototypic member of this family and consists of 50 kDa (p50, NF-kB-1, KBF1, EBP-1) and 65 kDa (RelA) subunits. c-Rel is a primarily lymphoid restricted member of this family and participates in the latter stages of B-cell differentiation (6-8). Both c-Rel and p65 heterodimerize with p50 (5). NF-id3/Rel proteins bind to a sequence element in the intronic enhancer of the kappa light chain gene (5'-GGGACTT'1 CC-3') (Ig-iB) and activate kappa light chain gene expression (9). NF-KB/Rel proteins are sequestered in an inactive state within cytoplasmic complexes. Cellular stimulation by cytokines, mitogens and pathogens, etc. induce the dissolution of inhibitory cytoplasmic complexes and the nuclear translocation of NF-KB/Rel proteins (reviewed in 5). IDEMSA can be used to determine the protein composition of protein-DNA complexes, their distribution, and relative abundance (Fig. 1 A). Nuclear or cytoplasmic extracts are depleted of either c-Rel, RelA, or p50 by incubation with the relevant antibody and protein A-Sepharose. The depleted extracts are then analyzed for the presence of c-Rel, RelA, and p50 by the EMSA/supershift assay. The combined results of immunodepletion and supershifts determine the protein composition of a particular protein-DNA complex and the localization of the dimer to a specific complex. Nuclear extracts were prepared from the pre-B-cell line 70Z/3 (kind gift from Dr Carol Sibley, University of Washington, *

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Seattle, WA) after 24 h of LPS stimulation (10 gg/ml Salmonella typhosa W0901, Difco, Detroit, MI) (10). Extracts (180 jig) were adjusted to 1 mg/ml in EMSA binding buffer [ 12 mM HEPES, pH 7.3,4 mM Tris-Cl, pH 7.5, 100 mM KCI, 1 mM EDTA, 20 mM DTT, 1 mg/ml BSA (fraction V, protease-free, Boehringer Mannheim, Indianapolis, IN), 0.167 mg/ml poly d[I-C], Boehringer Mannheim, Indianapolis, IN]. Where possible, samples were diluted to a final concentration of 1 mg/ml. Otherwise, the samples were adjusted to EMSA binding conditions with as minimal dilution as possible. Standard EMSA binding reaction was performed (10). Following binding, the sample was divided into four equivalent portions for either mock, c-Rel, RelA, or p50 depletion. Each portion, except the mock depleted control, received 3 jig of either the c-Rel(C)X, p65(C-20)X, or pSONLSX antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and 0.2 mM PMSF. The samples were incubated at 4°C overnight. The following morning, the samples were layered onto protein A-Sepharose pellets recovered by centrifugation (500 g for 5 min at 4°C) from 50 jl of a 10% slurry (in PBS plus 2% BSA and 0.02% azide). The samples were gently rocked on a rotary platform at 4°C for 30 min then centrifuged (500 g for 5 min at 4°C). The supernatants were transferred to a fresh tube with a Hamilton syringe, and incubated with 2 jig of antibodies for 1 h at 4°C. Following incubation, the antibody complexes were removed by protein A-Sepharose as described above. The supernatants were further purified by a final round of protein A-Sepharose absorption (a pellet from 25 jl of a 10% slurry) for 15 min at 4°C. Each immunodepleted sample, except the mock depleted control, was divided into four equivalent portions, with each portion containing -10 jig of extract. Supershift assays were then performed, under EMSA binding buffer conditions, by adding 2 jig of either the Rel(C)X, p65(C-20)X, or p5ONLSX antibodies to three of the four tubes in 20-25 jl volumes (