CelLytic PN Extraction Kit

Figure 2. Gel Shift analysis of protein-DNA complex formed between CREB DNA binding site and CREB protein in Tobacco Nuclear extracts. Protein extracts were prepared with the CelLytic-PN kit from nuclei isolated at three levels of purity (high, semi-pure, crude). A double stranded ³²P labeled CREB oligonucleotide probe was incubated with 1 µg of cytoplasmic or nuclear extract. Binding reactions with the nuclear extracts were performed in the absence [-] of competitor oligonucleotide (lanes 1-5) or in the presence of x100 or x500 fold excess of unlabeled CREB binding motif oligonucleotide (specific competitor [SP], lane 6 and lane 7, respectively) or in the presence of x100 fold excess of unlabeled oligonucleotide (non specific competitor, [NS] lane 8).
Lane 1: Free probe without extract.
Lane 2: Cytoplasmic fraction present in the supernatant of "High" level of purity.
Lane 3: Nuclear proteins isolated by "High" level of purity.
Lane 4: Nuclear proteins isolated by "Semi-pure" level of purity.
Lanes 5-8: Nuclear proteins isolated by "Crude" level of purity.
Binding reactions were run on a non-denaturing 6% polyacrylamide gel, dried and imaged on film. The arrows indicate the CREB-DNA complex and the free probe.