CRISPR-Cas9 Antibody Summary
Accession # Q99ZW2
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Detection of Human CRISPR-Cas9 by Simple WesternTM. Simple Western lane view shows lysates of HEK293T human embryonic kidney cell line either mock transfected or transfected with Cas9, loaded at 0.2 mg/mL. A specific band was detected for CRISPR-Cas9 at approximately 150 kDa (as indicated) using 20 µg/mL of Mouse Anti-CRISPR-Cas9 Monoclonal Antibody (Catalog # MAB10252). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Detection of CRISPR-Cas9 by Western Blot. Western blot shows lysates of HEK293T human embryonic kidney cell line either mock transfected or transfected with CRISPR-Cas9. PVDF membrane was probed with 2 µg/mL of Mouse Anti-CRISPR-Cas9 Monoclonal Antibody (Catalog # MAB10252) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for CRISPR-Cas9 at approximately 160 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Preparation and Storage
- 12 months from date of receipt, -20 to -70 °C as supplied.
- 1 month, 2 to 8 °C under sterile conditions after reconstitution.
- 6 months, -20 to -70 °C under sterile conditions after reconstitution.
Streptococcus pyogenes Cas9 (CRISPR associated protein 9) is a 160 kDa RNA guided endonuclease that introduces site specific cleavage of double strand DNA (1). It is part of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system found in many bacteria such as S. pyogenes and most archaea, which provide adaptive immunity against invading mobile genetic elements (such as viruses, transposable elements and conjugative plasmids) (2, 3). Upon viral infection, short viral DNA (known as "spacers") integrate into the host genome between CRISPR repeats, and RNA sequences (guide RNA or gRNA) with this genetic information help guide Cas9 protein to recognize and cut foreign DNA. Cas9 protein undergoes conformational changes upon gRNA binding that shift from non-DNA binding conformation into an active DNA binding conformation. In the Cas9-gRNA complex, the gRNA sequence remains accessible to interact with free DNA, and the extent to which the gRNA spacer and target DNA segment (known as "protospacer") match will determine the cut site (4). The presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of protospacers is required for Cas9 cleavage of the foreign DNA. PAM is absent in bacterial CRISPR loci, therefore preventing cleavage of the host genome (4). Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM specificity to this process (5). This RNA guided nuclease system called CRISPR/Cas (CRISPR associated protein) has been widely applied to genome engineering with increased efficiency (6). The attached nuclear localization signals(NLSs) on the chimeric protein ensures nuclear compartmentalization in mammalian cells during gene editing (7).
- Feng, Z. et al. (2013) Cell 154:1380.
- Moineau. et al. (2010) Nature 468:67.
- Barrangou, R. et al. (2014) Molecular Cell 54:234.
- Charpentier, E. et al. (2011) Nature 471:602.
- Leler, R. et al. (2015) Nature 519:199.
- Thomson, J.A. et al. (2013) PNAS,110:15644.
- Cong, L. et al. (2013) Science 339:819.
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