Overview

  • Product name

  • Description

    Rabbit polyclonal to CRISPR-Cas9
  • Host species

    Rabbit
  • Specificity

    This antibody reacts with CRISPR-Cas9, this antibody has been found to react with 6xHis and Flag tags. If using multiple tags we recommend ab189380.

  • Tested applications

    Suitable for: ICC/IF, IP, Flow Cyt, WBmore details
  • Species reactivity

    Reacts with: Streptococcus pyogenes
  • Immunogen

    Recombinant fragment corresponding to Streptococcus pyogenes CRISPR-Cas9 (N terminal). The immunogen was tagged with a 6xHis tag and an S tag this antibody has been found to react with 6xHis and Flag tags.
    Database link: Q99ZW2

  • Positive control

    • WB: HEK-293T cells transfected with DDDDK tag-CRISPR-Cas9 lysate. ICC/IF: U-2 OS cells transfected with DDDDK tag-CRISPR-Cas9. Flow Cyt: HEK-293T cells transfected with DDDDK tag-CRISPR-Cas9. IP: CRISPR-Cas9 IP in U-2 OS cells.
  • General notes

    Older lots react with cas9 and the Flag and 6x His tags.

    New lots react with only cas9  and the 6x His tag.

    Please contact our scientific support team for clarification.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.05% Sodium azide
  • Purity

    Whole antiserum
  • Clonality

    Polyclonal
  • Isotype

    IgG

Applications

Our Abpromise guarantee covers the use of ab204448 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF 1/1000 - 1/10000.
IP Use at an assay dependent concentration.

Use 10µl per IP.

Flow Cyt 1/1000 - 1/5000.
WB 1/1000 - 1/2000. Predicted molecular weight: 158 kDa.

Target

  • Relevance

    [FUNCTION] CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) (Probable). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.
  • Database links

    • Alternative names

      • Cas9 antibody
      • CRISPR-associated endonuclease Cas9/Csn1 antibody
      • CRISPR-Cas9/Csn1 antibody
      • csn1 antibody
      • SpyCas9 antibody
      see all

    Images

    • Lanes 1-2 : Anti-DDDDK tag antibody
      Lanes 3-4 : Anti-CRISPR-Cas9 antibody (ab204448) at 1/2000 dilution

      Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
      Lanes 2 & 4 : HEK-293T cells transfected with DDDDK tag-CRISPR-Cas9 lysate
      Lane 3 : HEK-293T cell lysate

      Lysates/proteins at 40 µg per lane.

      Predicted band size: 158 kDa



      WB: Transfer in nitrocelullose, block: 1 hour 5% milk in TBST (Tween= 0.2%),

      Incubation with primary antibody o/n at 4°C in 5% milk TBST (Tween= 0.2%).

    • Immunofluorescent analysis of U-2 OS (Human bone osteosarcoma epithelial cell line) cells transfected with DDDDK tag-CRISPR-Cas9, labeling CRISPR-Cas9 with ab204448 at 1/10,000 dilution (green). Labeling with anti-DDDDK tag (red). Right hand panel shows merged image with DAPI staining (blue).

      Protocol: Fixation with PFA 2% 10 min, permeabilization cold methanol, block with BSA 2% for 1h at RT, incubation with ab204448 1/10,000 1h 30 min at RT.

       

      There is a low background staining with ab204448 which is minimized at 1/10000 dilution.

    • Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells (upper panel), and transfected with CRISPR-Cas9 (lower panel) labeling CRISPR-Cas9 with ab204448 at 1/5000 dilution.

      Cells were fixed with 1% PFA and incubated with the primary antibody (1/5000) at RT for 2 hours.

    • Western blot analysis of immunoprecipitate using ab204448 in U-2 OS (Human bone osteosarcoma epithelial cell line) cells extracts.

      WB: anti-DDDDK tag.


      Lane 1: Input.
      Lane 2: Immunoprecipitation with control serum (10µl).
      Lane 3: Immunoprecipitation with ab204448 (10µl)

    References

    This product has been referenced in:

    • Martin AS  et al. A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Sci Rep 9:497 (2019). WB . Read more (PubMed: 30679582) »
    • Ajina R  et al. SpCas9-expression by tumor cells can cause T cell-dependent tumor rejection in immunocompetent mice. Oncoimmunology 8:e1577127 (2019). WB . Read more (PubMed: 31069138) »
    See all 9 Publications for this product

    Customer reviews and Q&As

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cells)
    Gel Running Conditions
    Reduced Denaturing (4-12% gradient bis tris)
    Loading amount
    10 µg
    Treatment
    1ug/ml Doxcycline
    Specification
    HeLa cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

    Abcam user community

    Verified customer

    Submitted Nov 08 2018

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