Immunofluorescent analysis of Ubiquitin in U2OS cells using a Ubiquitin Recombinant Rabbit Multiclonal Antibody (ab277768) followed by detection using an Alexa Fluor^® 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI(Image B) and actin stained with Alexa Fluor^® 594 phalloidin (red) (image C). Image D is a composite image showing cytoplasmic localization of Ubiquitin.

Anti-Ubiquitin antibody [10HCLC] (ab277768) at 2 µg/ml + K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell extract

Predicted band size: 26 kDa

Chromatin immunoprecipitation analysis of Ubiquitin was performed using cross-linked chromatin from 1 x 10⁶ HCT116 human colon carcinoma cells treated with serum for 0, 15, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 μL/100 μL well volume of a Ubiquitin Recombinant Rabbit Multiclonal Antibody (ab277768). Chromatin aliquots from ~1 x 10⁵ cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 μL of eluted DNA in 2 μL SYBR real-time PCR reactions containing primers to amplify -3.2kb upstream of the human FOS gene, or exon-4 of human FOS. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the FOS locus is shown above the data where boxes represent exons (grey boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by FOS primers are represented by black bars.