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Gel Shift (EMSA) Protocol |
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Credit: Dr. Mirmira, http://faculty.virginia.edu/mirmira/ There are multiple variations to this protocol, but we find that this one works well in all cases we tested. Reagents: 5X EMSA Buffer: 50mM HEPES (pH 7.9) 375 mM KCl 12.5 mM MgCl2 0.5 mM EDTA 5 mM DTT 15% Ficoll 32P-labeled oligonucleotide probe polydI/dC: 1 mg/ml in TE BSA: 10 mg/ml in TE 5% Polyacrylamide gel (30 ml) 22 ml water 3 ml 5X TBE 5 ml 30% Acrylamide 0.210 ml 10% Ammonium persulfate 10-100 ml TEMED 5X TBE (1 L): 54 g Tris base 27.5 g boric acid 20 ml 0.5 M EDTA (pH 8.0) Reaction: Combine the components in the following order (in ml): 5X EMSA buffer: 4 Water: to a final volume of 20 ml PolydI/dC: 1 BSA: 1 32P probe: 1 (10K cpm) protein: 1-3 ml let the reaction stand for 10-15 min at room temp., then load 18 ml per lane on a 5% polyacrylamide gel. Run at 150 V for 2h at room temperature, then dry the gel and expose 4-16 h to film at –80 C. |
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