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We compare the results using either a fluorescently labeled or radiolabeled DNA probe in the Gel Shift Assay System (Cat.# E3300) to detect DNA binding activity of AP1 and HeLa Nuclear Extract. |
Introduction
The gel shift, or electrophoretic mobility shift, assay provides a simple and rapid method for detecting DNA-binding proteins. This method has been used widely in the study of sequence-specific DNA-binding proteins such as transcription factors. The assay is based on the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The gel shift assay is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or whole cell extract preparations), with a labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences.
Gel shift assays typically use 32P-labeled DNA probes. In this experiment, we have used an AP1 oligonucleotide labeled at the 5´-ends with the fluorescent dye, carboxytetramethylrhodamine (TMR) as a nonradioactive alternative.
Methods
DNA Probe Preparation: The AP1 Consensus Oligonucleotide supplied with the Gel Shift Assay System (Cat.# E3300) was 5´-end labeled with [γ-32P]-ATP and T4 Polynucleotide Kinase (Cat.# M4101) following the procedure in Technical Bulletin #TB110. The oligonucleotide was purified using a NICK® column (Pharmacia). Approximately 8 picomoles of labeled oligonucleotide was eluted in 400µl 1xTE buffer; 50,000-100,000cpm/µl). For TMR-labeled AP1 oligonucleotides, complementary strands were synthesized with TMR at the 5´-end of the top strand. The two strands were annealed, then verified to be double-stranded by polyacrylamide gel analysis (final concentration = 2pmol/µl).
Gel Shift Analysis: Gel shift analysis was performed using the Gel Shift Assay System. Gel shift reactions were assembled in a total volume of 10µl containing 1X Gel Shift Binding Buffer, 1µl labeled AP1 Consensus Oligo in the presence or absence of a source of AP1 protein (either human recombinant AP1 (0.5fpu/µl; Cat.# E3061) or HeLa Nuclear Extract included with the Gel Shift Assay System), and in the presence or absence of cold AP1 Consensus Oligo (1–3µl; 2pmol/µl). The assembled reactions were incubated on ice for 20-30 minutes, then each reaction was combined with 1.5µl Gel Shift Loading Dye (250mM Tris-HCl, [pH 7.5], 0.2% bromophenol blue, 40% glycerol) and loaded onto a 6% DNA retardation gel (Novex) to which current had been applied for 10 minutes at 300 volts. The gels were run for 15–20 minutes at 300 volts in 0.5X TBE. The gels were then either exposed to film (radioactive probe) or scanned on the Hitachi FMBIO® II using the 585 channel (fluorescent probe).
Results
Figure 1 shows a typical gel shift assay using a radioactive oligo probe. The 32P-labeled AP1 Oligonucleotide bound to both the recombinant human AP1 and to protein in the HeLa extract. Cold AP1 Oligonucleotide added to the gel shift reactions competed for binding with the labeled AP1 Oligonucleotide, reducing the intensity of the band. Gel shift assays using TMR-labeled AP1 Oligonucleotide produced results that were similar to the experiments using radiolabeled probe (Figure 2). Binding activity was easily detected using 1µl of the rhAP1 or HeLa Extract.
Conclusions
Fluorescent detection of DNA-protein complexes provides a nonradioactive alternative to the typical 32P-labeld gel shift assay. Oligonucleotide probes can be synthesized with a fluorophore label at the 5´-end. TMR was used in this experiment, while other researchers have successfully used carboxyfluorescein (FAM) or BODIPY® 630/650 (1).
Reference
Figures for Non-Radioactive Gel Shift Assays
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Figure 1. Gel shift analysis using radiolabeled AP1 Consensus Oligonucleotide. Reactions were performed as described in Technical Bulletin #TB110 using 32P-labeled AP1 Consensus Oligonucleotide (and either 1µl recombinant human AP1 (Cat.# E3061; Panel A) or 1µl HeLa Nuclear Extract (Panel B) in the presence (+) or absence (–) of competing cold AP1 Oligonucleotide. A reaction without a source of AP1 protein was performed as a control. |
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Figure 2. Gel shift analysis using TMR-labeled AP1 Consensus Oligonucleotide. Reactions were performed as described in Technical Bulletin #TB110 using TMR-labeled AP1 Consensus Oligonucleotide and varying amounts of either recombinant human AP1 (Cat.# E3061; Panel A) or HeLa Nuclear Extract (Panel B). A reaction without a source of binding protein was performed as a control. |
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