CRISPR-Cas9 Antibody - BSA Free

Novus Biologicals | Catalog # NBP2-52718

Novus Biologicals
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Key Product Details

Species Reactivity

S. pyogenes

Applications

Western Blot, ELISA, Immunocytochemistry/ Immunofluorescence

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all CRISPR-Cas9 Primary Antibodies »

Product Specifications

Immunogen

CRISPR-Cas9 Antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to a region near the C-terminal of Streptococcus pyogenes CRISPR-Cas9 protein. (Uniprot: Q99ZW2)

Reactivity Notes

Reactive against CRISPR-Cas9 from Streptococcus pyogenes.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Description

The product was affinity purified from monospecific antiserum by immunoaffinity chromatography.

Store vial at -20C prior to opening. Aliquot contents and freeze at -20C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use.

Scientific Data Images for CRISPR-Cas9 Antibody - BSA Free

Western Blot: CRISPR-Cas9 Antibody [NBP2-52718]

Western Blot: CRISPR-Cas9 Antibody [NBP2-52718]

Western Blot: CRISPR-Cas9 Antibody [NBP2-52718] - Lane 1: 293T non transfected cell lysate. Lane 2: 293T Cas9 over expressed cell lysate. Load: 15ug per lane. Primary Antibody cas9 used at 1ug/mL overnight at 4C. Secondary Antibody: goat anti-rabbit HRP at 1:40,000 for 30 min at room temp. Expected theoretical molecular weight: 158 kDa.
Immunofluorescence: CRISPR-Cas9 Antibody [NBP2-52718]

Immunofluorescence: CRISPR-Cas9 Antibody [NBP2-52718]

Immunofluorescence: CRISPR-Cas9 Antibody [NBP2-52718] - HeLa cells transfected with GFP+Cas9 or GFP+CD-3. Fixation: Formaldehyde fixed, permeabilized Triton X-100. Primary: Anti-Cas9 used at 1ug/mL. Secondary: Donkey anti-Rabbit at 1ug/mL, staining seen in red. Bottom images show GFP stain overlay

Applications for CRISPR-Cas9 Antibody - BSA Free

Application
Recommended Usage

ELISA

1:25000-1:30000

Immunocytochemistry/ Immunofluorescence

1ug/ml

Western Blot

1:1000
Application Notes
This affinity purified antibody has been tested for use in ELISA, Immunofluorescence, and Western blotting. Specific conditions for reactivity should be optimized by the end user. Expect a band ~158 kDa in size corresponding to Cas-9 protein by western blotting in the appropriate cell lysate or extract.

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2

Format

BSA Free

Preservative

0.01% Sodium Azide

Concentration

Please see the vial label for concentration. If unlisted please contact technical services.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at -20C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.

Background: CRISPR-Cas9

Clustered regularly interspaced short palindromic repeats (CRISPRs) are derived from DNA fragments of bacteriophages that infect prokaryotes. When infected, the bacteria capture snips of DNA from the invading virus to create CRISPR arrays. During subsequent infections, the bacteria produce RNA segments from the CRISPR arrays to target the virus' DNA. CRISPR-associated protein 9 (Cas9) is RNA-guided, binds DNA, and is a cleaving enzyme that functions as an integral component of the bacterial CRISPR adaptive immune system that targets the virus' DNA to disable it (1). To check for sites complementary to the 20 base pair spacer region of the guide RNA (gRNA) of the CRISPR, Cas9 unwinds foreign DNA that invades the bacteria. If the DNA substrate is complementary to the gRNA, Cas9 cleaves the invading DNA, rendering the virus disabled. The presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the target DNA (protospacer) is required for Cas9 cleavage of foreign DNA. As PAM is absent in bacterial CRISPR loci, cleavage of the host genome is avoided and provides a novel sequence for identification of foreign DNA by Cas9.

Using CRISPR-Cas9 technology, double-stranded DNA breaks may be induced within specific targeted genome sequences (target DNA; protospacer) for insertion or removal of DNA sequences for gene editing applications. To target a specific loci, a gRNA that will bind to a specific target sequence of DNA within a genome is created. The gRNA will recognize the DNA sequence, and the Cas9 enzyme will cleave the DNA at the targeted location. Once the targeted DNA is removed by Cas9, the cell's own DNA repair mechanism is used to insert or remove a DNA sequence for genomic editing.

Cas9 detection is used to confirm and evaluate CRISPR Cas9 gRNA transfection efficiency. Western blot analysis of CRISPR-Cas9 gRNA transfected cell lysates with Cas9 antibodies identifies the protein having a theoretical molecular weight of 160kDa. Broad areas of research are benefiting from CRISPR-Cas9 based gene editing tools including studies of basic immunity functions, genetic screening and disease treatment (2). Ethical concerns have led to many countries making it illegal to manipulate human germline cells or perform embryo genome editing.

References

1. Oakes, B. L., Fellmann, C., Rishi, H., Taylor, K. L., Ren, S. M., Nadler, D. C.,... Savage, D. F. (2019). CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification. Cell, 176(1-2), 254-267.e216. doi:10.1016/j.cell.2018.11.052

2. Chiou, S. H., Winters, I. P., Wang, J., Naranjo, S., Dudgeon, C., Tamburini, F. B.,... Winslow, M. M. (2015). Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev, 29(14), 1576-1585. doi:10.1101/gad.264861.115

Long Name

CRISPR-associated Protein 9

Alternate Names

Cas9, CRISPR-associated endonuclease Cas9/Csn1, CRISPR-Cas9/Csn1, CRISPR/Cas9, csn1, SPy_1046, SPy1046, SpyCas9, CRISPR-associated protein 9 nuclease

UniProt

Q99ZW2

Additional CRISPR-Cas9 Products

Product Documents for CRISPR-Cas9 Antibody - BSA Free

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Product Specific Notices for CRISPR-Cas9 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

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