A micro-scale method to isolate DNA-binding proteins suitable for quantitative comparison of expression levels from transfected cells.

While evaluating methods for assessing quantitative levels of transcription factor-I (TEF-1) expression in transfected cells, we found that current micro-scale protocols (1,2) were unsuitable for comparing accurately the expression of DNA binding proteins among different cell lines or transfected cells. Here we report a rapid, one-step protocol utilizing whole cell extracts, which is appropriate for detection and quantitative analysis of DNA binding activities from small numbers ofcells. This procedure has been found to be ideal for the evaluation of upand downmodulation of transcriptional factors in transfected cells. We found that the hypotonic lysis step of current micro-scale procedures to isolate DNA-binding proteins resulted in significant loss ofDNA binding activities (Fig. lA, compare DIGNAM and HYPO). Consequently, we examined direct, high salt extraction of DNA-binding proteins from whole cells utilizing a buffer containing high potassium salt. For comparison of the different protocols (Fig. lA), extracts from -108 BeWo cells were prepared by the Dignam et al. (4), Manley et al. (5) or the protocol described below. The hypotonic lysate from the Dignam protocol was concentrated by lyophilization and dialyzed against Dignam buffer D. The amount of extract used in each case (Fig. IA) was adjusted to that representing 2 x 105 cells. Figure 1B shows results from three different transfected cell lines. For these studies, 5 x 106 cells were electroporated in the presence of 5 ,ug plasmid DNA (3) and plated onto 10 cm dishes. After 20 h the cells were rinsed twice with cold phosphate buffered saline (PBS), harvested by scraping with a plastic policeman in 1.5 ml PBS and transferred into 1.5 ml microcentrifuge tubes. Following centrifugation at 800 g for 1 min, the PBS was removed by aspiration. Cell pellets were resuspended with vortexing at room temperature in three packed cell volumes of extraction buffer (20 mM HEPES-HCl pH 7.9,20% glycerol, 0.5M KCl, 0.2mM EDTA, 0.5 mM PMSF and 0.5 mM DTT). To achieve complete lysis, the samples were subjected to one cycle of freezing on dry ice and immediately thawing at 37'C in a water bath. Increasing the number of freeze-thaw cycles did not increase the yield of DNA-binding activities (Fig. IA). Also, inclusion of the nonionic detergent, NP-40, in any of the protocols did not improve significantly the efficiency ofDNA-binding protein activity (Fig. IA). The samples were then centrifuged at 14 000 g for 30 s and the supernatant was diluted with 4 vol of dilution buffer (extraction buffer lacking KCl) to reduce the salt concentration to that of Dignam Buffer D [(4), 20 mM HEPES-HCl pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT]. The extracts were immediately stored at -70°C until use and have been found to be viable for periods up to 1 year. The exact protocol worked equally well with 5 x 105 to _108 cells. Extracts prepared by the current protocol contained more TEF-1 binding activity than extract prepared by the Dignam (4) or Manley (5) procedures. Also, significant TEF-1 binding activity was found in the hypotonic supernatants (4) of BeWo cells (Fig. 1A), indicating that leakage from the nuclear compartment occurs during this step. Thus omission ofhypotonic lysis may improve the yield ofDNA-binding activity and provide for more consistent extraction of activities when quantitative comparison is desired. The use of 0.5 M KCl in the extraction buffer followed by dilution to 0.1 M KC1 allowed the extracts to be used directly in gel shift and footprinting assays. Transfectionmediated expression of TEF1 in three different cell lines using extracts isolated by this protocol could be readily distinguished from the variable levels of endogenous TEF-1 (Fig. IB). Comparable results have also been obtained in expression experiments with Spi, a relatively abundant transcription factor, the pituitary specific factor, GHF-1 and the estrogen receptor (not shown). Since the protein-DNA binding is usually carried out in 0.5x Dignam Buffer D (4), up to half of reaction volume can be derived from the extract. For the factors we measured, clearly visible signals were observed using 5-10 pl of each extract (-10-20% of the total). Since electroporation results in a large proportion of cells which are non-viable and do not plate, < 5 x 106 cells might be used with alternate transfection approaches. Due to the simplicity of the method, extract preparation from multiple samples is highly reproducible (data not shown). Application of this method to functional studies of TEF-1 expression in BeWo cells using both over-expression and anti-sense oligonucleotide approaches have confirmed the appropriate upor down-modulation of TEF expression using gel shift assays and Western blot analyses (6).