Antibody was affinity purified using an epitope specific to ASH2 immobilized on solid support. The epitope recognized by A300-489A maps to a region between residue 575 and the C-terminus (residue 628) of human Absent, Small, or Homeotic-Like 2 using the numbering given in Swiss-Prot entry Q9UBL3 (GeneID 9070). Immunoglobulin concentration was determined by extinction coefficient: absorbance at 280 nm of 1.4 equals 1.0 mg of IgG.
ASH2, ash2 (absent, small, or homeotic)-like, ASH2L1, ASH2L2, ASH2-like protein, Bre2, set1/Ash2 histone methyltransferase complex subunit ASH2
between 575 and C-term
2 - 8°C
Detection of human ASH2 by western blot and immunoprecipitation. Samples: A. Whole cell lysate from HEK293T cells that were mock transfected (E, 50 µg) or transfected with ASH2 expression constructs containing HA-tagged ASH2 (H, 25 µg) or Flag-tagged ASH2 (F, 25 µg). B. Whole cell lysate from one 10cm plate of normal 293T cells (~1 mg protein; 1/2 of IP loaded/lane). Antibodies: Affinity purified rabbit anti-ASH2 antibody A300-489A used at 1 µg/ml for WB (A and B) and at 5 µg/plate for IP. ASH2 was also immunoprecipitated with rabbit anti-ASH2 antibody A300-107A using 5 µg/plate. Detection: Chemiluminescence with an exposure time of 1 second (A and B).
ChIP-chip scatter plot of anti-Ash2 enriched DNA binding sites versus input reference DNA. A. 10 µg of A300-489A was used to immunoprecipitate chromatin from K-562 cells according to Ren et al (Genes Dev. 2002 16: 245-256). immunoprecipitatesd DNA and reference DNA were amplified via ligation-mediated PCR and the products labeled with fluorescent dNTPs. The labeled ChIP and reference DNA were pooled, hybridized to a DNA microarray, and analyzed. Data points below the +3 SD curve (red line) represent significantly enriched binding sites. B. As a control, a similar experiment was performed using normal rabbit IgG. Compared to the anti-Ash2 ChIP, normal rabbit IgG showed little enrichment.