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. Author manuscript; available in PMC: 2009 Mar 5.
Published in final edited form as: J Biol Chem. 2004 Apr 15;279(27):28209–28219. doi: 10.1074/jbc.M403553200

Fig. 2. Affinity purified DNA-binding proteins.

Fig. 2

A, purification of NF-κB DNA-binding proteins by NF-κB oligonucleotide DNA affinity chromatography. Various fractions obtained from the NF-κB affinity column were subjected to 10% SDS-polyacrylamide gel electrophoresis and thereafter visualized by silver staining. Lane 1 represents the crude nuclear extract; lanes 2 and 3 represent the first flow-through and first wash flow-through with 800 mM KCl containing binding buffer, respectively. Lanes 4–8 represent the wash flow-through with 1x binding buffer. Lane 9 is 150 mM KCl in 1x binding buffer wash flow-through, and lane 10 is 450 mM KCl in 1x binding buffer elute. B, EMSA of affinity purified NF-κB DNA-binding protein complexes. For the supershift assay, anti-p50 or anti-p65 antibodies of 1 μg were preincubated with the EMSA reaction mixture, and the reaction was performed as described under “Experimental Procedures.” DNA binding complexes and supershift complexes are indicated by the arrow. C, Western blot analysis of affinity purified DNA-binding proteins. 450 mM KCl, eluted from an affinity column that contained purified protein complexes, was subjected to 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, and then probed with either p50 or p65 antibody.