Key features and details
- Mouse monoclonal [7A9-3A3] to CRISPR-Cas9
- Suitable for: ICC/IF, WB
- Reacts with: Streptococcus pyogenes
- Isotype: IgG1
Product nameAnti-CRISPR-Cas9 antibody [7A9-3A3]
See all CRISPR-Cas9 primary antibodies
DescriptionMouse monoclonal [7A9-3A3] to CRISPR-Cas9
Tested applicationsSuitable for: ICC/IF, WBmore details
Species reactivityReacts with: Streptococcus pyogenes
Recombinant fragment corresponding to Streptococcus pyogenes CRISPR-Cas9 (N terminal).
- WB: S2 cells transfected with CRISPR-Cas9 ICC-IF: NIH/3T3-Cas9 transfected cells.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C long term.
Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
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...
PurityProtein G purified
Light chain typekappa
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.
|ICC/IF||Use a concentration of 5 - 10 µg/ml.|
|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.
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.
- Cas9 antibody
- CRISPR-associated endonuclease Cas9/Csn1 antibody
- CRISPR-Cas9/Csn1 antibody
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.
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.
Lane 1: HeLa inducible Cas9 cell line in the absence of Doxcycline (-DOX)
Lane 2: HeLa inducible Cas9 cell line in the treated for 24 hours in the presence of 1ug/ml of Doxcycline (+DOX).
Blot was probed with Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) 1:2000 and Anti-Tubulin Antibody DM1A (Sigma) as loading control. Shows specific band at 170kDA in Dox treated cells and tubulin band at 50kDa. Lysis and running Cells lysed in 1.5X Lammeli buffer +0.15M DTT syringed 10X with 25g needle and then boiled 100 degrees C for 10 minutes. 10ug of sample run out on 4-12% Bis Tris Nupage gel in 1X MOPS buffer WB protocol Western blot was performed by wet transfer of gel onto nitrocellulose membrane 100V for 2 h 4 degrees. Blocked in 5% milk PBST for 1 h room temp (1X PBS + 0.1% tween 20) incubated 24h at 4 degrees in 5% PBST with primary antibodies 1:2000 Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) as well as 1:3000 Anti Tubulin Mouse monoclonal DM1A antibody ( Sigma) Washed 3X PBST IgG anti mouse HRP 1:5000 (Jackson immunoscience) Washed 3x PBST Washed
HeLa cell line contains Cas9 under the control of a Doxcycline/Tetracycline inducible promotor (Tet on) Specific band at 170 kDa appears upon treatment with Doxcycline 1ug/ml for 24hours.1X PBS Developed using ECL prime kit (GE healthcare).
ab191468 has been referenced in 25 publications.
- Li K et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat Commun 11:485 (2020). PubMed: 31980609
- Qiu W et al. Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system. Nucleic Acids Res 47:e52 (2019). PubMed: 30805613
- Zuo Z et al. Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain. Elife 8:N/A (2019). PubMed: 31361218
- Josipovic G et al. Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system. Nucleic Acids Res 47:9637-9657 (2019). PubMed: 31410472
- 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). PubMed: 30858603