Figure 2.

Purification of CRSP and identification of CRSP polypeptides. (a) Reconstituted transcription reactions with the GC box template, Sp1 supplemented with nuclear extract (NE) (lane 1), or the P1M and RNA polymerase II and basal transcription factor fraction (lane 2). The rest of the reactions contained GC box template, Sp1, RNA polymerase II and basal transcription factors, and IP-TFIID (lanes 3–14), supplemented with Ni²⁺-NTA-agarose flow-through (FT) and eluate (El) (lanes 3 and 4), Cibacron Blue Sepharose (B) FT and El (lanes 5 and 6), and various fractions derived from Poros Heparin (lanes 7–14). (b) Reconstituted transcription reactions with GC box template, Sp1, RNA polymerase II and basal factor fraction, and IP-TFIID (lanes 1–11) supplemented with input and fractions derived from glycerol gradient centrifugation (lanes 1–5) or input and fractions derived from MonoS chromatography (lanes 6–11). (c) Silver-stained SDS/PAGE gel of the MonoS purified CRSP. Molecular size standards are shown on the left, and the apparent molecular weight of each polypeptide is listed on the right.