LSD1 Antibody - ChIP-seq Grade

Figure 1. ChIP results obtained with the Diagenode antibody directed against LSD1
ChIP was performed with the Diagenode antibody against LSD1 (Cat. No. C15410067) on sheared chromatin from 4,000,000 K562 cells using the “iDeal ChIP-seq” kit (Cat. No. C01010055).. An antibody titration consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for specific regions in the MYT1, RBM19, and TGFBR3 genes, used as positive controls, and for the MYOD1 gene, used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against LSD1
ChIP was performed on sheared chromatin from 4,000,000 K562 cells using 1 μg of the Diagenode antibody against LSD1 (cat. No. C15410067) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. 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 peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure 2A and B) and in three regions surrounding the MYT1, RBM19 and TGFBR3 positive control genes, respectively (figure 2C, D and E). The position of the amplicon used for ChIP-qPCR is indicated by an arrow.

Figure 3. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against LSD1 (Cat. No. C15410067) in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:176,000.

Figure 4. Western blot analysis using the Diagenode antibody directed against LSD1
Western blot was performed using nuclear extracts from HeLa cells (40 μg) and the Diagenode antibody against LSD1 (Cat. No. C15410067) diluted 1:4,000 in TBS- Tween containing 5% skimmed milk. The molecular weight marker (in kDa) is shown on the left. The location of the protein of interest is indicated on the right.

Figure 5. Western blot analysis using the Diagenode antibody directed against LSD1
Whole cell extracts (40 μg) from HeLa cells transfected with LSD1 siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the Diagenode antibody against LSD1 (Cat. No. C15410067) diluted 1:5,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Figure 6. Immunofluorescence using the Diagenode antibody directed against LSD1
HeLa cells were stained with the Diagenode antibody against LSD1 (Cat. No. C15410067) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the LSD1 antibody (left) diluted 1:200 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.