Diagenode

CRISPR/Cas9 polyclonal antibody

Catalog Number
Format
Price
C15310258-100
100 μl
$490.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the Cas9 nuclease (CRISPR-associated protein 9) using a recombinant protein. 

LotA2508-001
ConcentrationNot determined
Species reactivityStreptococcus pyogenes
TypePolyclonal ChIP-grade / ChIP-seq grade
PurityWhole antiserum
HostRabbit
Storage ConditionsStore at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.
PrecautionsThis 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

* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 µl per IP.

TESTIMONIAL

I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.

ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.

Researcher from the University of Manchester
  • Validation data

    ChIP

    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).

    A.

    B.

    C.

    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.

    WB

    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.

    IP

    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.

    IF

    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.

  • Background

    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.

  •  Testimonials

    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
  •  Applications
    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
  •  Documents
    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
  •  Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: CRISPR/Cas9 polyclonal antibody (Diagenode Cat# C15310258-100 Lot# A2508-001). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    (Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes
    Neguembor M.V. et al.
    CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISP...

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