CRISPR/Cas9 polyclonal antibody

Diagenode’s CRISPR/Cas9 polyclonal antibody shows superior signal than the original clone 7A9: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.

Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.

Lot	A2508-004
Concentration	Not determined
Species reactivity	Streptococcus pyogenes
Type	Polyclonal, ChIP grade, ChIP-seq grade
Purity	Whole antiserum from rabbit containing 0.05% azide.
Host	Rabbit
Storage Conditions	Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.
Precautions	This product is for research use only. Not for use in diagnostic or therapeutic procedures.

Applications	Suggested dilution	References
ChIP/ChIP-seq ^*	2-5 µl/ChIP	Fig 1, 2
Western Blotting	1:5,000	Fig 3
Immunoprecipitation	1 µl/IP	Fig 4
Immunofluorescence	1:1,000	Fig 5

Figure 1. ChIP using the Diagenode antibody directed against Cas9
ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, 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 Cas9
ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. 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 in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.

Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9
Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.

Figure 4. IP using the Diagenode 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 counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.

CRISPR systems are adaptable immune mechanisms which are present in many bacteria to protect themselves from foreign nucleic acids, such as viruses, transposable elements or plasmids. Recently, the CRISPR/Cas9 (CRISPR-associated protein 9 nuclease, UniProtKB/Swiss-Prot entry Q99ZW2) system from S. pyogenes has been adapted for inducing sequence-specific double stranded breaks and targeted genome editing. This system is unique and flexible due to its dependence on RNA as the moiety that targets the nuclease to a desired DNA sequence and can be used induce indel mutations, specific sequence replacements or insertions and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate upregulation of specific endogenous genes or to alter histone modifications or DNA methylation.

Diagenode’s CRISPR/Cas9 polyclonal antibody shows superior signal than the original clone 7A9: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.

Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.
EPFL in Lausanne

IP
Immunoprecipitation Read more

IF
Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more

WB
Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more

ChIP-qPCR (ab)
Read more

C15310258-Datasheet CRISPR Cas9 Polyclonal antibody DATASHEET Datasheet description	Download
Accurate QC to optimize CRISPR/Cas9 genome editing specificity POSTER The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease res...	Download
Optimize the selection of guide RNA by ChIP to keep CRISPR on-target APPLICATION NOTE The mechanisms of target recognition and target specificity of the Cas9 protein is still not comp...	Download

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CRISPR/Cas9 polyclonal antibody

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