Anti-RING1 | E3 ubiquitin-protein ligase RING1

Western Blot (WB)

Figure 1. Western Blot (WB) result.

20 µg of total protein from Psc/Su(z)2-KO cells (Kahn et al., 2016. doi: 10.1093/nar/gkw701 , line1) and Ras3 (wild type, line2) cells lysed with 1x SDS page loading buffer were separated on 12% SDS-PAGE and blotted 2h to PVDF using tank transfer. Blot was dried and incubated in the primary antibody at a dilution of 1: 2000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice in 1x PBS. Blot was incubated in secondary antibody (anti-rabbit IgG AP conjugated, from Promega) diluted to 1:10 000 in for 30 minutes at RT with agitation. The blot was washed as above and developed with NBT/BCIP solution (SIGMA).

Courtesy of Dr. Alexander Glotov. , Umeå University, Sweden


Chromatin Ummunoprecipitation (ChIP)

Figure 2. ChIP recovery.

ChIP and qPCR analysis were done as described [Schwartz YB, Kahn TG, Nix DA, Li XY, Bourgon R, et al. (2006) Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat Genet 38: 700–705. doi: 10.1038/ng1817]. Chromatin from Ras3 cells was used for ChIP. Quantitative PCR was performed with primers specific for BXD-PRE of Ubx gene (Polycomb target gene in repressed state), used as positive controls, and for intergenic region, used as negative control (NC). Figure 2 shows the ChIP recovery measured by qPCR as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA). Chromatin from 5x107 cells and 3 µg of anti-RING1 antibody were used for ChIP reaction.

Courtesy of Dr. Tatyana Khan, Umeå University, Sweden.