CRISPR-Cas9 Antibody - BSA Free

Novus Biologicals | Catalog # NBP2-66379

Novus Biologicals
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Key Product Details

Species Reactivity

Bacteria

Applications

Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all CRISPR-Cas9 Primary Antibodies »

Product Specifications

Immunogen

This CRISPR-Cas9 antibody was raised against full length S. pyogenes Cas9 protein [UniProt Q99ZW2]

Specificity

This CRISPR-Cas9 antibody is specific to Cas9 protein from Streptococcus pyogene serotype M1.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

158.4 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for CRISPR-Cas9 Antibody - BSA Free

Western Blot: CRISPR-Cas9 AntibodyBSA Free [NBP2-66379]

Western Blot: CRISPR-Cas9 AntibodyBSA Free [NBP2-66379]

Western Blot: CRISPR-Cas9 Antibody [NBP2-66379] - Total protein from Cas9 transfected Hek293T cells was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/ml anti-Cas9 in block buffer and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Expected molecular weight: 158.4 kDa.

Applications for CRISPR-Cas9 Antibody - BSA Free

Application
Recommended Usage

Western Blot

2 ug/ml

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

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

Stability & Storage

Store at 4C short term. Aliquot and store at -20C 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, CRISPR-associated protein 9 nuclease

Additional CRISPR-Cas9 Products

Product Documents for CRISPR-Cas9 Antibody - BSA Free

Certificate of Analysis

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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

View specific protocols for CRISPR-Cas9 Antibody - BSA Free (NBP2-66379):

CRISPR-Cas9 Antibody:
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute anti-CRISPR-Cas9 primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.

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