Application

Detect Cas9 D10A/H840A nickase mutant using this rabbit polyclonal Anti-Cas9, D10A/H840A Mutant, Cat. No. ABC1490, validated for use in Immunoprecipitation and Western Blotting.

Research CategorySecondary & Control Antibodies

Immunoprecipitation Analysis: 10 µg from a representative lot immunoprecipitated exogenously expressed Cas9 nickase D10A mutant (with C-terminal Myc-DDK tag) from 500 µg lysate of transfected HEK293 cells.

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

CRISPR-associated endonuclease Cas9/Csn1 (UniProt Q99ZW2; also known as SpyCas9) is encoded by the Streptococcus pyogenes serotype M1 gene Cas9 (also known as csn1) gene (Gene ID 901176). Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA loci containing short repetitions of base sequences complementary to antecedent mobile elements and target invading nucleic acids. Each repetition is followed by short segments of spacer DNA from previous exposures to a virus. Cas9 is an essential endonuclease in Streptococcus pyogenes serotype M1′s CRISPR immune system that confers resistance to foreign genetic elements such as plasmids and phages. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9. 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, and then trimmed 3′-5′ exonucleolytically. Mutations of the catalytic residues (D10A in RuvC and H840A in HNH) convert Cas9 into DNA nickases (Cas9n) that cleave only the strand complementary to the crRNA, minimizing off-target activity.

Immunogen

A mixture of two KLH-conjugated peptides corresponding to the Cas9 target sites sequences with D10A and H840A mutations.

Legal Information

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Other Notes

Concentration: Please refer to lot specific datasheet.

Physical form

Purified rabbit polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Affinity purified.

Quality

Evaluated by Western Blotting in lysate from HEK293 transfectants expressing Cas9 nickase D10A.

Western Blotting Analysis: 2 µg/mL of this antibody detected exogenously expressed Cas9 nickase D10A mutant (with C-terminal Myc-DDK tag) in 10 µg of lysate from transfected HEK293 cells.

Specificity

This polyclonal antibody will detect Cas9 with D10A mutation in the RuvC-I region and/or H840A mutation in the HNH domain (e.g. AKA60242.1), but not Cas9 with neither D10A nor H840A mutation (e.g. NP_269215).

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Target description

~160 kDa observed. 158.3 kDa calculated (based on AKA60242.1). Uncharacterized bands may be observed in some lysate(s).