Anti rabbit igg cy5

Whole-cell extracts were prepared by freeze and thaw lysis (three cycles) in 600 mM NaCl, 20 mM Tris-HCl pH 7.8, 20% glycerol. After SDS–PAGE, proteins were transferred onto PVDF membrane in semi-dry conditions. The membrane was then blocked in 5% non-fat dry milk buffer and incubated with mouse anti-γH2AX (Clone JBW301, Upstate, 1:5,000). Immunoblots were stained with corresponding HRP-conjugated secondary antibodies (GE Healthcare, 1:20,000) and detected with the enhanced chemiluminescence detection system (Amersham Biosciences). Quantification was performed using ImageJ.
For the validation of antibody specificity and cross-reactivity, a dilution series of synthetic peptides (CKATQASQEY; Peptide Specialty Laboratories GmbH), with the underlined serine in either phosphorylated or non-phosphorylated form, was immobilized on a nitrocellulose membrane at the indicated concentrations and probed with anti-γH2AX and anti-H2AX as described above.
CTCF knockdown western blots were developed using a rabbit anti-CTCF (#D31H2, Cell Signaling, 1:700) and a mouse anti-actin (AC-40, Sigma-Aldrich, 1:1,000) and overnight incubation at 4 °C, followed by a direct immunofluorescence detection using anti-rabbit-IgG-Cy5 (#711-175-152, Jackson, 1:1,000) and an anti-mouse-IgG-Alexa488 (A11029, Invitrogen, 1:1,000). Images were recorded using a AI600 Imager (Amersham) and quantified using ImageJ.

Rabbit polyclonal antiserum was raised against bacterially expressed polypeptides carrying the C-terminal region of Pc, the N-terminal region of E(z) or the N-terminal region of Pho. Immunoblot data using these antibodies are shown in Fig. S6. Immunostaining of imaginal discs was carried out as described previously (Liu et al., 2003 (link)). Antibodies were used at the following dilutions: anti-Pc (1:1000), anti-E(z) (1:1000), anti-Pho (1:1000), anti-Ubx (Developmental Studies Hybridoma Bank; 1:1000), anti-Mbf1 (Jindra et al., 2004 (link); 1:500), goat anti-rabbit IgG or anti-mouse IgG Alexa488 (Molecular Probes, A72731 and A32723; 1:2000) and anti-rabbit IgG-Cy5 (Jackson ImmunoResearch, 111-225-144; 1:500). Images were acquired with an LSM510 META confocal microscope (Zeiss).

HaCaT and HaSKpwC7 cells were harvested and lysed in RIPA buffer. Protein concentration was determined by 660 nm Protein Assay (Pierce). Indicated amounts were loaded. After separation on a 12% PAA-SDS gel, the bands were visualized using mouse-anti-actin (clone AC-40, 1:2,000) and rabbit-anti-p53 (Cell signalling, #9282, 1:1,000) followed by anti-mouse-IgG-Cy3 and anti-rabbit-IgG-Cy5 (both Jackson ImmunoResearch, 1:3,000).