AbFlex® Cas9 antibody (rAb)

RRID: AB_2793783
Aliases: CRISPR/Cas9
Catalog No: 91123 Format: 100 µg $350 Buy
Catalog No: 91124 Format: 10 µg $75 Buy

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Antibody Type:
Recombinant
Isotype:
IgG2a
Purification:
Ni-NTA
Host:
Mouse
Molecular Weight:
160 kDa
Reactivity:
Human

Applications

ChIP Validated ChIP-Seq Validated Western Blot Validated Enzyme-linked Immunosorbent Assay Validated

Application Notes

Applications Validated by Active Motif:
ChIP-Seq: 4 µg per ChIP
Bead-based ELISA: 9 - 75 ng/ml
WB: 2 µg/ml

AbFlex® recombinant antibodies are genetically derived from DNA sequences of parental hybridoma clones. For details on the parental clone, see Catalog No. 61577.

Immunogen

This antibody was raised against a recombinant protein within the N-terminal region of Streptococcus pyogene Cas9. This antibody recognizes Cas9 and dCas9.

Buffer

Purified IgG in 200 mM Hepes pH 7.5, 100 mm NaCl, 50 mM NaOAc, 30% glycerol and 0.035% sodium azide.

 

AbFlex® Cas9 antibody (rAb) tested by Western blot.
50 ng recombinant Cas9 protein was probed with 0.5 µg/ml AbFlex® Cas9 antibody (Lane 1). HEK293T cells were transfected with a plasmid containing a mammalian expression construct for dCas9 (S. pyogenes). Chromatin was prepared 48 hours post-transfection, boiled and 15 µl (~200,000 cell equivalents) was run on a SDS-PAGE gel and probed with 2 µg/ml AbFlex® Cas9 antibody (Lane 2). A molecular weight marker was run in Lane 3.

AbFlex® Cas9 antibody (rAb) tested by bead-based specificity analysis.
Luminex bead-based specificity analysis was used to confirm the specificity of AbFlex® Cas9 antibody (rAb) antibody for Cas9. Various proteins were conjugated to MagPlex Luminex beads and incubated with various amounts of AbFlex® Cas9 antibody (rAb). Protein-bound antibody was detected with anti-mouse IgG-Phycoerythrin and read in a Luminex instrument.

Luminex® is a registered trademark of Luminex Corporation.

AbFlex® Cas9 recombinant antibody (rAb) tested by ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT® High Sensitivity Kit (Cat. No. 53040) with 30 µg of Jurkat cell chromatin and 4 µg of AbFlex Cas9 antibody. ChIP DNA was sequenced on the Illumina NextSeq and 8 million sequence tags were mapped to identify binding sites on chromosome 4.

Background

AbFlex® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6xHis Tag, a Biotinylation Tag for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports.

AbFlex® Cas9 antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells.

Cas9 is a nuclease from Streptococcus pyogenes that can be targeted to particular DNA sequences through a guide RNA that results in double-stranded breaks in DNA. Cas9 is part of the CRISPR/Cas9 gene-editing system that can create a DNA break at a specific location with the genome.

CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) Probable. In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently 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, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.

Chromatin IP Assays

This antibody has been validated for use in ChIP and/or ChIP-Seq, and can be used with Active Motif's ChIP-IT® High Sensitivity Kit or our magnetic bead-based ChIP-IT® Express Kits. For an overview of all of our ChIP products, please visit our Chromatin IP Page to learn about ChIP products designed for use with AbFlex® Cas9 antibody (rAb).

Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20°C for up to 2 years. Keep all reagents on ice when not in storage.

Guarantee

This product is guaranteed for 12 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • ChIP = ChIP Sequencing;
  • CUT&RUN = Cleavage Under Targets and Release Using Nuclease;
  • CUT&Tag = Cleavage Under Targets and Tagmentation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • NEU = Neutralizing;
  • WB = Western Blotting