Description: This 6H4 monoclonal antibody recognizes Staphylococcus aureus Cas9 endonuclease.
Applications Reported: This 6H4 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This 6H4 antibody has been tested by intracellular staining followed by flow cytometric analysis of Staphylococcus aureus derived Cas9-GFP transfected 293T cells using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Please refer to "Staining Intracellular Antigens for Flow Cytometry, Protocol A: Two step protocol for intracellular (cytoplasmic) proteins" located at www.thermofisher.com/flowprotocols. This may be used at less than or equal to 0.06 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.
Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222-49) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-57) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser
Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in bacteria and genetic engineering. The system is unique and flexible due to its dependence on RNA as the element that targets the nuclease to a desired DNA sequence. It can be used to induce indel mutations, specific sequence replacements/insertions, and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate upregulation of specific endogenous genes or to alter histone modifications or DNA methylation. Cas9 may be derived from Streptococcus pyogenes (SpCas9) or Staphylococcus aureus (SaCas9). SaCas9 has a number of properties that make it advantageous for genome editing, including its small size, high efficiency, nickase activity, and apparent specificity. SaCas9 principally recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at rates comparable to SpCas9 with an ideal spacer lengths of 24 to 20 nucleotides. In comparison to SpCas9, SaCas9 is smaller (1368 AA versus 1053 AA) and generates indels at a comparable rate when directed against target DNA with the mutually permissive NGGRRT PAM. This makes it the preferred enzyme for all-in-one delivery of Cas9 and multiple gRNA expression cassettes with Adeno-associated vectors.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: cas9; CASP 9; Caspase-9 precursor; CASPASE-9c; Caspase9; CRISPR-Cas9; CRISPR9; CTD; OTTHUMP00000002324; RNCASP9; RP11-265F14.3; s aureus