HEK293 cells were transfected with a construct including the N-terminal 608 amino acids of <em>S. pyogenes</em> CAS9 fused to GFP and stained with mouse mAb to N-terminal region of <em>S. pyogenes</em> Cas9, MCA-3F9, in red. Transfected cells express the green fluorescent fusion protein and bind the antibody in red, producing a yellow signal. Nuclear DNA in transfected and non-transfected cells is revealed with the blue DNA stain Hoechst.
HEK293 cells were transfected with a construct including the N-terminal 608 amino acids of <em>S. pyogenes</em> CAS9 fused to GFP and stained with mouse mAb to N-terminal region of <em>S. pyogenes</em> Cas9, MCA-3F9, in red. Transfected cells express the green fluorescent fusion protein and bind the antibody in red, producing a yellow signal. Nuclear DNA in transfected and non-transfected cells is revealed with the blue DNA stain Hoechst.

EnCor Biotechnology

Mouse Monoclonal Antibody to S. pyogenes CAS9 Cat# MCA-3F9

$250.00
Description

      The MCA-3F9 antibody was made against the N-terminal region of S. pyogenes CAS9. It can be used to verify the expression of S. pyogenes CAS9 on blots, in cells and tissues. The same immunogen was used to generate a rabbit polyclonal antibody to CAS9, RPCA-CAS9-Sp. EnCor also manufactures antibodies against the CAS9 homologue from S. aureus.

Amount: 100µL of 1mg/mL
Amount: 100µL of 1mg/mL
HEK293 cells were transfected with a construct including the N-terminal 608 amino acids of S. pyogenes CAS9 fused to GFP and stained with mouse mAb to N-terminal region of S. pyogenes Cas9, MCA-3F9, in red. Transfected cells express the green fluorescent fusion protein and bind the antibody in red, producing a yellow signal. Nuclear DNA in transfected and non-transfected cells is revealed with the blue DNA stain Hoechst.
Western blot analysis of HEK293 cell lysates using mouse mAb to S. pyogenes CAS9, MCA-3F9: [1] protein standard (red), [2] transfected cells with S. pyogenes CAS9 (N-terminal 1-680 amino acids) construct, [3] non-transfected cells, and [4] full length recombinant S. pyogenes CAS9 protein.

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Name: CAS9 from S. pyogenes, mouse monoclonal antibody, Cat# MCA-3F9
Immunogen: N-terminal region, amino acids 1-608 of Cas9 sequence CDJ55032.1 from Streptococcus pyogenes, expressed in and purified from E. coli.
HGNC Name: NA, no human homolog
UniProt: Q99ZW2
Molecular Weight: 160kDa
Host: Mouse
Isotype: IgG1 heavy, κ light
Species Cross-Reactivity: Streptococcus pyogenes
RRID: AB_2858267
Format: Purified antibody at 1mg/mL in 50% PBS, 50% glycerol plus 5mM NaN3
Applications: WB, IF/ICC
Recommended Dilutions: WB: 1:1,000-1: 2,000 on CAS9 transfected cells and 1:10,000-20,000 on pure full length CAS9 protein. IF/ICC:1:1,000-2,000. IHC not tested
Storage: Store at 4°C for short term, for longer term at -20°C.

      A recent revolution in biology has been stimulated by the discovery of CRISPR, or “Clustered Regularly Interspaced Short Palindromic Repeats” and the understanding of the "CRISPR Associated" enzymes (CAS refs 1,2). The CRISPR repeated sequences are found in bacterial genomes and function as part of unique bacterial immune system which contain short DNA sequences derived from viruses which have infected the bacteria. These virally derived sequences can make short RNA sequences which can hybridize with specific viral DNA and target a nuclease, such as CAS9, to the viral sequence. So CAS9 is directed to cleave the specific viral sequence and so inactivate the virus. The RNA sequence can be designed to specifically cut DNA virtually anywhere, including in the genomes of living human and other mammalian cells, allowing inexpensive gene editing with unprecedented ease. For example three groups of researchers essentially cured the disease state in a mouse model of Duchenne muscular dystrophy (3-5). A similar approach essentially cured dogs affected with a related disease state (6). Several varieties of CAS9 have been studied and there are several other related enzymes with similar properties. Much of the early work was performed with CAS9 from Streptococcus pyogenes which is rather large at ~158kDa, so the corresponding DNA is also rather large at about 4.2kb. This is problematic with some expression systems especially since DNA encoding RNA sequences and possibly other regulatory elements are usually required. The CAS9 gene of Staphylococcus aureus is significantly smaller, 3kb, producing a protein of 124kDa (7). For an excellent recent review of the various CAS family enzymes and their utility see reference 8.

      This antibody has not been tested on formalin fixed and paraffin embedded samples for IHC, and so cannot be recommended for this purpose.

1. Hsu PD, Lander ES, Zhang F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157:1262-78 (2014).
2. Doudna1 JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9 Science 346:1077-86 (2014)
3. Long C, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351:400-3 (2015).
4. Nelson CE, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403-7 (2015).
5. Tabebordbar M, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:407-11 (2015).
6. Amoasii L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.
Science doi:10.1126/science.aau1549 (2018).
7. Ran FA, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186-91 (2015).
8. Knott GJ, Doudna J. CRISPR-Cas guides the future of genetic engineering. Science 361:866-9 (2018).

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