Figure 1.

Affinity purification of Gro-interacting proteins. (A) Silver-stained SDS–polyacrylamide gel showing results of a small scale affinity purification of Gro-interacting proteins. (Lane 1) Molecular mass markers; (lane 2) unfractionated 0- to 12-hr Drosophila embryonic nuclear extract; (lanes 3–6) SDS elutions of M₂ antibody beads lacking (M₂, lanes 3,5) or containing (M₂Gro, lanes 4,6) epitope-tagged Gro. (Lanes 5,6) The beads were incubated with embryonic nuclear extract and subsequently subjected to extensive washing prior to SDS elution; (lanes 3,4) the nuclear extract was omitted. IgG light (L) and heavy (H) chains are indicated. Polypeptides from the embryonic nuclear extracts that uniquely coprecipitate with the M₂Gro beads are indicated (p140, p110, p68, p38, and p31). (*) Two polypeptides that are retained on the beads in both the presence and absence of Gro. (B) Silver-stained SDS–polyacrylamide gel showing an aliquot of the eluate from a large-scale M₂Gro affinity column used for affinity purification of Gro-interacting proteins. Elution was achieved by washing the beads with excess Flag peptide. Thus, unlike in A, the immunoglobulin chains were not eluted. However, the same two nonspecific polypeptides (*) that eluted with SDS (A), eluted with the peptide, indicating that they are probably binding directly to the antibody. The amino acid sequence denotes a peptide sequence obtained from an internal lysC fragment of p68. This sequence is a perfect match to a predicted lysC fragment in Drosophila histone deacetylase, Rpd3. P38 has been identified as Drosophila histone H1 (H1). (C) Immunblot with anti-Rpd3 antibody. Lanes are the same as in A.