CRISPR/Cas9 polyclonal antibody

Figure 1. ChIP using the Diagenode antibody directed against Cas9
ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50µg chromatin was incubated overnight at 4°C with either 5 µg of an anti-FLAG antibody or 2 µl of the Diagenode antibody against Cas9 (Cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. Figure 5 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

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Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9
ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and either an H19 or a GAPDH sgRNA cells using 5 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) and the iDeal ChIP-seq kit for transcription factors (C01010055). The IP'd DNA was subsequently analysed on an Illumina HiSeq 3000. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile of the H19 and GAPDH transfected cells in a region of chromosome 11 surrounding the H19 gene (fig 2A) of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak obtained with the GAPDH sgRNA in the YIPF4 gene.

Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9
Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.

Figure 4. IP using the Diagenode monoclonal antibody directed against Cas9
IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.

Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9
HeLa cells expressing Cas9 under the control of the tight TRE promoter were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.