Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468)

Overview

  • Product name

    Anti-CRISPR-Cas9 antibody [7A9-3A3]
    See all CRISPR-Cas9 primary antibodies
  • Description

    Mouse monoclonal [7A9-3A3] to CRISPR-Cas9
  • Host species

    Mouse
  • Tested applications

    Suitable for: ICC/IF, IHC - Wholemount, WBmore details
  • Species reactivity

    Reacts with: Streptococcus pyogenes
  • Immunogen

    Recombinant fragment corresponding to Streptococcus pyogenes CRISPR-Cas9 (N terminal).

  • Positive control

    • WB: S2 cells transfected with CRISPR-Cas9 ICC-IF: NIH/3T3-Cas9 transfected cells.
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C long term.
  • Storage buffer

    pH: 7.4
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Some batches contain 6.97% L-Arginine as a stabilizing agent. For lot-specific buffer information, please contact our Scientific Support team.
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Monoclonal
  • Clone number

    7A9-3A3
  • Isotype

    IgG1
  • Light chain type

    kappa

Applications

Our Abpromise guarantee covers the use of ab191468 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 Use a concentration of 5 - 10 µg/ml.
IHC - Wholemount 1/500. PubMed: 27638686
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 160 kDa (predicted molecular weight: 160 kDa).

We recommend using 3% milk as the blocking agent for Western blot.

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

    • All lanes : Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) at 5 µg/ml

      Lane 1 : S2 non-transfected cell lysate
      Lane 2 : S2 cells transfected with CRISPR-Cas9 plasmid

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 160 kDa
      Observed band size: 160 kDa


      Exposure time: 4 minutes


      We recommend using 3% milk as the blocking agent in Western Blot.

    • ab191468 stained in NIH3T3 cells. Untreated and Cas9 transfected cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab191468 at 10µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab150117 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    References

    This product has been referenced in:

    • Park H  et al. In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease. Nat Neurosci 22:524-528 (2019). Read more (PubMed: 30858603) »
    • Simhadri VL  et al. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev 10:105-112 (2018). Read more (PubMed: 30073181) »
    See all 16 Publications for this product

    Customer reviews and Q&As

    1-10 of 12 Abreviews or Q&A

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (eHAP)
    Permeabilization
    Yes - 0.1% TritonX-100
    Specification
    eHAP
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 15% · Temperature: RT°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Sep 14 2016

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    No
    Specification
    HeLa
    Blocking step
    BSA as blocking agent for 15 minute(s) · Concentration: 1% · Temperature: 21°C
    Fixative
    Methanol

    Abcam user community

    Verified customer

    Submitted Nov 19 2018

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa (stably expressing doxycycline-inducible cas9)
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris)
    Loading amount
    15 µg
    Treatment
    1 µg/mL Doxacycline (Lane 2)
    Specification
    HeLa (stably expressing doxycycline-inducible cas9
    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 16 2018

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cell line with Tet inducible Cas9)
    Gel Running Conditions
    Reduced Denaturing (4-12% Tris/Bis Nupage gel)
    Loading amount
    10 µg
    Treatment
    Doxcycline 1um/ml for 24h
    Specification
    HeLa cell line with Tet inducible Cas9
    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 07 2018

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain, all cell types)
    Permeabilization
    Yes - Triton 0.1%
    Specification
    Brain, all cell types
    Blocking step
    Casein as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
    Fixative
    Paraformaldehyde

    Mrs. Susana González Granero

    Verified customer

    Submitted Sep 13 2018

    Answer

    Yes, the antibody will detect the Cas9 mutated form D10A. There is a  review of the antibody at this link, https://www.abcam.com/CRISPR-Cas9-antibody-7A9-3A3-ab191468/reviews/46701, which includes a western blot showing detection of Cas9 D10A in lane #3.

    Read More
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (C2C12)
    Gel Running Conditions
    Reduced Denaturing (4-12% Tris-Bis Nupage gel)
    Loading amount
    10 µg
    Specification
    C2C12
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

    Abcam user community

    Verified customer

    Submitted Dec 12 2017

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (C2C12)
    Permeabilization
    Yes - 0.4% triton X-100
    Specification
    C2C12
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 12 2017

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK293)
    Gel Running Conditions
    Reduced Denaturing (7.5%)
    Loading amount
    20 µg
    Specification
    HEK293
    Blocking step
    Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

    Dr. Biswajyoti Sahu

    Verified customer

    Submitted Jul 25 2016

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Breast and lung)
    Gel Running Conditions
    Reduced Denaturing (Gradient)
    Loading amount
    40 µg
    Specification
    Breast and lung
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jan 12 2016

    1-10 of 12 Abreviews or Q&A

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