Journal:

Article Title: Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein

doi:

Figure Lengend Snippet: Purification scheme of coactivators and TFIID. Starting material for the nuclear extract material was between 60 and 120 liters of cultured cells. Arrows indicate subsequent columns used for purification. Columns shown with horizontal bars were developed with step elutions in buffer with the salt concentrations indicated. Columns shown with slanted bars represent linear gradients with initial and final salt concentrations indicated at the left and right. Semipurified TFIID is the DE-52 0.25 M KCl peak. Purified TFIID eluted from the PureGel SCX column at approximately 150 mM KCl. The coactivator present in the DE-52 0.1 M KCl flowthrough was purified by two separate methods. Purification over a PureGel SCX column yielded the mixed coactivator peak. Purification by gel filtration yielded three activities, LTF1, LTF2, and STF.

Article Snippet: The complementary activity flowed through this column, was diluted with HE 0 to reduce the final KCl concentration to 50 mM, and was applied to a PureGel SCX (strong cation-exchange) column (Rainin).

Techniques: Purification, Cell Culture, Filtration

Journal:

Article Title: Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein

doi:

Figure Lengend Snippet: Purification of LTF2. (A) The various stages of purification of LTF2 were analyzed by SDS-PAGE and visualized by silver staining. The following fractions that contained the peak of LTF2 activity are in the lanes as indicated: 0.3 to 0.5 M KCl step of the phosphocellulose column; 0.1 M KCl flowthrough of the DE-52 column; fractions corresponding to molecular weights of approximately 80 to 130 kDa of the Superdex 200 column; 0.7 to 0.8 M ammonium sulfate fraction of a linear gradient on the phenyl-Superose column; 0.25 to 0.3 M KPO4 of a linear gradient on the hydroxyapatite column; and the 150 to 180 mM KCl fractions of a linear gradient on an analytical 4.6-mm by 10-cm PureGel SCX column. Molecular masses are indicated (in kilodaltons) on the left. (B) Highly purified PARP from the PureGel SCX column peak shown in panel A were substituted for LTF2 in transcription reactions using conditions similar to those in Fig. ​Fig.5.5. STF, PARP, and LTF1 were added as indicated. The arrow indicates the full-length transcript. (C) Transcripts were quantitated as in Fig. ​Fig.44.

Article Snippet: The complementary activity flowed through this column, was diluted with HE 0 to reduce the final KCl concentration to 50 mM, and was applied to a PureGel SCX (strong cation-exchange) column (Rainin).

Techniques: Purification, SDS Page, Silver Staining, Activity Assay