Anti-CRISPR/Cas9 antibody, Mouse monoclonal
SIGMA/SAB4200701 - clone 7A9-3A3, purified from hybridoma cell culture
Synonym: CRISPR-associated protein-9 nuclease; Crisper; Crispr RNA
Product Type: Chemical
Immunofluorescence Human HEK-293T cells overexpressing CRISPR/Cas9 were fixed and permeabilized with 4% paraformaldehyde followed by 0.5% Triton X-100. Fixed cells were stained with 2 μg/mL Mouse Anti-CRISPR/Cas9, Clone: 7A9-3A3 (Cat. No. SAB4200701). The antibody was developed using Goat Anti-Mouse IgG, Cy3 conjugate. Cells were counterstained with DAPI (blue) to stain nuclei (merged).
Immunofluorescence Human HEK-293T cells overexpressing CRISPR/Cas9 were fixed and permeabilized with 4% paraformaldehyde followed by 0.5% Triton X-100. Fixed cells were stained with 2 μg/mL Mouse Anti-CRISPR/Cas9, Clone: 7A9-3A3 (Cat. No. SAB4200701). The antibody was developed using Goat Anti-Mouse IgG, Cy3 conjugate. Cells were counterstained with DAPI (blue) to stain nuclei (merged).
Western Blotting Recombinant CRISPR proteins expressed in E. coli were separated on SDS-PAGE (0.5 μg/lane) and probed with 1 μg/mL Anti-CRISPR/cas9 antibody, Mouse monoclonal, Clone: 7A9-3A3 (Cat. No. SAB4200701). The antibody was developed using Goat Anti-Mouse IgG-Peroxidase (Cat. No. A2304) and a chemiluminescent substrate. Lanes 1. SpCas9 protein 2. Cas9 nickase protein 3. Dead Cas9 (dCas9) protein 4. FnCas9 protein 5. AsCpf1 protein 6. LbCpf1 protein
Immunoprecipitation Mouse Anti-CRISPR/Cas9, Clone: 7A9-3A3 (Cat. No. SAB4200701) was used to immunoprecipitate CRISPR/Cas9 from human HEK-293T cells overexpressing CRISPR/Cas9. Detection antibody: Mouse Anti-CRISPR/Cas9 (Cat. No. SAB4200701), followed by Goat Anti-Mouse IgG-Peroxidase (Cat. No. A2304) and a chemiluminescent substrate. Lanes 1. Negative control: without lysate 2. Negative control: without antibody 3. 5 μg/test antibody 4. Positive control: 50 μL total cell lysate of HEK-293T cells overexpressing CRISPR/Cas9 (1:10 ratio from total cell lysate used for IP)
| antibody form | purified from hybridoma cell culture |
| antibody product type | primary antibodies |
| biological source | mouse |
| clone | 7A9-3A3, monoclonal |
| concentration | ~1 mg/mL |
| form | buffered aqueous solution |
| isotype | IgG1 |
| packaging | antibody small pack of 25 μL |
| Quality Level | 200  |
| shipped in | dry ice |
| storage temp. | −20°C |
| target post-translational modification | unmodified |
| technique(s) | immunoblotting: 1-2 μg/mL using whole extracts of human HEK-293T cells over-expressing CAS9 protein |
| immunofluorescence: 1-2 μg/mL using human HEK-293T cells over-expressing CAS9 protein. | |
| immunoprecipitation (IP): 5-10 μg using whole extract of human HEK-293T cells over-expressing CAS9 protein |
| Application: | Monoclonal Anti-CRISPR/CAS9 recognizes over-expression systems containing CAS9 construct. The antibody successfully recognizes mutant Cas9 variants, including nickase Cas9 and dead Cas9 (dCas9). The antibody may be used in various immunochemical techniques including Immunoblotting, Immunofluorescence and Immunoprecipitation. Monoclonal Anti-CRISPR/CAS9 does not cross react with FnCas9 from Francisella novicida bacteria and Cpf1 proteins from Acidaminococcus sp. (strain BV3L6) and Lachnospiraceae bacterium ND2006. |
| Biochem/physiol Actions: | CAS9 plays a vital role in plasmid DNA interference. It is the only cas protein needed to deliver resistance against foreign DNA. CAS9 stimulates both RNA-guided genome editing and gene regulation in various organisms, but it can facilitate only one activity at a time within any given cell. |
| Disclaimer: | Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. |
| General description: | CAS9, also known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease , cas5 and csn1, is the signature gene of the type II CRISPR -RuvC (RNase H-like fold) cas system. CAS9 contains 1388 amino acids. This protein is predicted to contain a RuvC/ ribonuclease (RNase) H domain involved in crRNA maturation and McrA/HNH signature domain involved in the DNA degradation step. |
| General description: | CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) belongs to the type II CRISPR/CAS9 system. It is part of an adaptive immune system of the Streptococcus pyogenes SF370, protecting from pathogens targets genes by cleaving the foreign DNA in a sequence-dependent manner.2 The type II CRISPR/Cas system which has been adapted to expression in eukaryotic cells, consists of four genes including the Cas9 (CRISPR-associated proteins) nuclease, two noncoding CRISPR RNAs (crRNAs, or gRNA), trans-activating crRNA (tracrRNA) and a precursor crRNA (pre-crRNA) array. The pre-crRNA contains nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). The Cas9 endonuclease can be engineered with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB. In comparison to other genome-editing technologies such as designer zinc fingers (ZFs), transcription activator–like effectors (TALEs) and homing meganucleases, the CRISPR/CAS9 system is a scalable, affordable and easy to engineer. Therefore, the anti-CRISPR/CAS9 antibody can be a useful tool for detecting CRISPR/CAS9 positively transfected cells, reveling DSB sites in the genome and in ChIP (Chromatin Immunoprecipitation) related assays. |
| Immunogen: | a recombinant protein within the N-terminal region of Streptococcus pyogene Cas9 |
| Other Notes: | This product is for R&D use only, not for drug, household, or other uses. In order to obtain best results in different techniques and preparations we recommend determining optimal working concentration by titration test. |
| Physical form: | Supplied as a solution in 0.01 M phosphate buffered saline pH 7.4, containing 15 mM sodium azide as a preservative. |
| Preparation Note: | For continuous use, store at 2-8°C for up to one month. For extended storage, freeze in working aliquots. Repeated freezing and thawing is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use. Working dilution samples should be discarded if not used within 12 hours. |
| RIDADR | NONH for all modes of transport |
| WGK Germany | WGK 1 |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | −20°C |
| UNSPSC | 12352203 |