Gel Mobility Shift Assay Conditions

400 microliters 50% glycerol100 mM Tris-HCl pH8 @ 25 degrees 100 microliters 1 M Tris pH8300 mM KCl 300 microliters 1 M KCl25 mM MgCl2 25 microliters 1 M MgCl2500 micrograms/ml BSA 5 microliters 100 mg/ml BSA 170 microliters H2Ooptional: add 25 microliters saturated bromophenyl blue [BioRad] (~0.1% in H2O) per ml of 5X buffer (this may inhibit the binding of some proteins)Store buffer at -70 degrees.Gel shift reactions are performed as follows: 20 microliter binding reaction:4 microliters 5X binding buffer0.2 microliters 0.1 M DTT2000-5000 cpm labeled DNA0.125 micrograms p[dG-dC]H2O to 20 microliters final volumeAdd proteins to reaction last. Incubate protein and DNA at room temperature for ~30-40 min and load to native gels which are run in the cold room at 4 degrees. Gels are not pre cooled but are set in cold room 5-10 minutes before loading and pre run at 160 V. The wells of the gels are rinsed out several times before prerunning and again before loading. Samples are applied to the gel while the gel is running. For best results, use a fine tip pipetman tip to load the gels. We run gels at 160V (12 cm long) for ~45 min. For a typical gel shift reaction (20 microliter reaction), I use 1-2 ng TBPc (the conserved region of yeast TBP from the Sigler lab) and and 5-10 ng of wild-type or truncated yeast TFIIB. The amount of proteins will have to be titrated for your specific conditions. Gels:10.5 ml (20%/0.33%) acrylamide/bis acrylamide3.5 ml 10X TGOE buffer1.75 ml 50% glycerol35 microliters 0.5 M DTT20.9 ml H200.3 ml 10% ammonium persulfate30 microliters TEMED 10X TG0E 500 ml0.25 M Tris 15.1 g Tris1.9 M glycine71.3 g glycinepH 8.3 with acetic acid at room temp. Adjust volume to 500 ml. Running buffer is 1X TGOE buffer
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