ChIP assays were performed using Kasumi-1 cells, the antibody against AML1-ETO and optimized primer pairs for qPCR. Sheared chromatin from 1.25 million cells and 4 ul of antibody were used per ChIP experiment. QPCR was performed using primers specific for the FUT7, NFE2 and OGG1 genes. Image shows the occupancy, calculated as the ratio + control/background for which the promoter of the H2B gene was used.
Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against AML1-ETO. The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:32, 750.
a) ChIP-seq results obtained with the ab against AML1-ETO ChIPÂ¡Â¯s was pooled and analysed with an Illumina Genome Analyzer. The 32 bp tags were aligned to the human reference genome (hg18) using the ELAND algorithm. Image shows the results of the complete chromosome 3 and three genomic regions surrounding the OGG1, FUT7 and NFE2 genes, respectively. The position of the PCR amplicon is indicated with an arrow.