Cas9 from Streptococcus pyogenes, mouse monoclonal, cat#MCA-3F9

Cas9 from Streptococcus pyogenes, mouse monoclonal, cat#MCA-3F9

Transfected Hek293 cells overexpressing the N-terminal 1-608 amino acids of S. pyogenes Cas9 . The cells were stained with MCA-3F9 in red, and these cells also appear yellow since we counterstained with our rabbit antibody RPCA-Cas9-AP  to the same construct in green, giving a yellow color. The N-terminal construct contains a nuclear localization sequence and so is predominantly nuclear in localization. Most Hek293 cells in this field are not transfected so only the nuclei of these cells can be visualized with the blue DAPI DNA stain.

Western blot analysis of MCA-3F9
Lane 1: Protein size marker with size in kiloDaltons..
Lane 2: Blot of crude protein extract from Hek293 culture transfected with the MCA-3F9 immunogen, the N-terminal 1-608 amino acids.
Lane 3: Non-transfected control Hek293 cell extract.
Lane 4: Blot of 40 ng Streptococcus pyogenes Cas9 was probed with MCA-3F9 at 1:1,000 dilution and, as expected, the antibody also recognizes full length Cas9 protein at 160 kDa.

Product name Anti- Cas9 from Streptococcus pyogenes
Description Mouse monoclonal to the N-terminal region (amino acid 1-608) of Cas9 from Streptococcus pyogenes
Reference Code MCA-3F9
HGNC name  NA, no human homolog
RRID# AB_2572246
Molecular weight 160kDa
Immunogen N-terminal region, amino acids 1-608 of Cas9 sequence CDJ55032.1  from Streptococcus pyogenes, expressed in and purified from E. coli.
Isotype IgG
Concentration Antibody is supplied as 100 µL purified material at 1 mg/mL diluted in PBS with 50% glycerol and 5mM sodium azide.
Applications Western blot, ICC/IF,
Suggestions for use Western blot: 1:1,000-1: 2,000 on Cas9 transfected cells and 1:10,000-20,000 on pure full length Cas9 protein. ICC/IF:1:1,000-2,000 .
Storage instructions Shipped on ice. Store at 4°C. For long term storage, keep at -20°C..

A recent revolution is biology has been stimulated by the discovery of CRISPR, or “Clustered Regularly Interspaced Short Palindromic Repeats” and the understanding of their significance. These repeated sequences are found in bacterial genomes and function as part of unique bacterial immune system. Interspaced between these repeated DNA sequences are 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, if the bacteria is infected by this virus again, Cas9 can be directed to cleave the specific viral sequence and so inactivate the virus. By careful design of the RNA sequence the system can be used to specifically cut DNA virtually anywhere, including in living human and other mammalian cells. This allows inexpensive gene editing with unprecedented ease, and much effort is going into refining the Cas9 enzymes and their relatives for use in mammalian systems. Recent papers in this exploding field showed that it is feasible to correct genetic defects in a variety of experimental situations. For example three groups of researchers essentially cured the disease state in a mouse model of Duchenne muscular dystrophy, a disease in which point mutations or frame shifts result in the production of a truncated and non-functional form of very large muscle protein dystrophin (1). This was performed using AAV vectors on adult animals, using RNA sequences which directed cleavage of the DNA at two sites flanking the genetic defect. The normal DNA repair mechanisms in some cases annealed the two cut sites effectively bypassing the defective region. This allowed the production of a slightly shorter but still functional dystrophin protein. There seems to be no reason why this approach would not work on humans also. Several varieties of Cas9 have been studied and there appear to be several other related enzymes with similar properties in bacteria. In particular the Cas9 homolog from Staphylococcus aureus is significantly smaller and so presents less problems when packaged into vectors (2). Much of the early work was performed with Cas9 from Streptococcus pyogenes. The S. pyogenes protein is rather large at 1,368 amino acids, ~160kDa, so the corresponding DNA is also rather large at about 4.2 kb. Our antibody is a mouse monoclonal raised against  amino acids 1-608 of Cas9 from S. pyogenes and binds the immunogen transfected into cells on western blots and in immunocytochemistry. The homologous region of the S. aureus Cas9 is not closely related in amino acid sequence and, as expected, this antibody does not recognize that protein. We also have monoclonal  antibody MCA-6F7 and polyclonal antibodies RPCA-Cas9-SA made in rabbit  and CPCA -Cas9-SA in chicken against Cas9 protein in S. aureus.


1. CRISPR helps heal mice with muscular dystrophy.
CRISPR helps heal mice with muscular dystrophy

2. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature 520:186-91 (2015).

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