The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent dilution. PubMed: 20237320
Use a concentration of 1 µg/ml. Detects a band of approximately 39 kDa (predicted molecular weight: 42,39 kDa).
Constitutes one of the E3 ubiquitin-protein ligases that mediate monoubiquitination of 'Lys-119' of histone H2A, thereby playing a central role in histone code and gene regulation. H2A 'Lys-119' ubiquitination gives a specific tag for epigenetic transcriptional repression and participates in X chromosome inactivation of female mammals. Essential component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex act via chromatin remodeling and modification of histones, rendering chromatin heritably changed in its expressibility. Compared to RNF2/RING2, it does not have the main E3 ubiquitin ligase activity on histone H2A, and it may rather act as a modulator of RNF2/RING2 activity.
Western blot - Anti-RING1 antibody - ChIP Grade (ab32644)
Lane 1: Wild type HAP1 whole cell lysate (20 µg) Lane 2: RING1 knockout HAP1 whole cell lysate (20 µg) Lane 3: MOLT4 whole cell lysate (20 µg) Lane 4: LnCaP whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32644 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32644 was shown to recognize RING1 in wild type cells as signal was lost at the expected MW in RING1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and RING1 knockout samples were subjected to SDS-PAGE. ab32644 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 2 µg/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.