CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free

Novus Biologicals | Catalog # NBP2-52398

Novus Biologicals
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Key Product Details

Validated by

Independent Antibodies, Biological Validation

Species Reactivity

Bacteria

Applications

Validated:

Western Blot, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation, Chromatin Immunoprecipitation (ChIP)

Cited:

Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # 6G12

Format

BSA Free
View all CRISPR-Cas9 Primary Antibodies »

Product Specifications

Immunogen

This CRISPR-Cas9 antibody (6G12) - C-terminus was raised against recombinant C-terminal fragment of S.pyogenes CRISPR/Cas9 (between amino acids 1139 to 1368) [UniProt# Q99ZW2]

Specificity

This CRISPR-Cas9 antibody (6G12) - C-terminus is specific to Cas9 from Streptococcus pyogene.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

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 (6G12) - C-terminus - BSA Free

Western Blot: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Western Blot: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Western Blot: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - Control HeLa cells (un-transfected) and HeLa cells expressing Flag-tagged S. pyogenes's CRISPR-Cas9 under the control of PTight (Tet-ON) promoter. Samples were treated for 24 hours with 1ug/uL of Doxycyclin and lysed under native conditions. 30 ug of the whole cell lysate from each sample type per lane was separated by 7.5% SDS-PAGE. Nitrocellulose membrane was incubated with CRISPR-Cas9 antibody clone 6G12 (hybridoma supernatant diluted 1:100 at 4C O/N). After washing, the membranes were incubated with secondary HRP-coupled antibody and bands were visualized by ECL and exposure of X-ray films. Prestained marker bands were visualized with Blue Marker Antibody (NBP2-33376). The image shown is from 1 minute exposure time. Observed molecular weight is ~158 kDa.
Immunocytochemistry/ Immunofluorescence: CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free [NBP2-52398]

Immunocytochemistry/ Immunofluorescence: CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free [NBP2-52398]

Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - HeLa cells or HeLa cells expressing Flag-tagged SpCas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/uL Doxycyclin, fixed and permeabilized with Methanol/Acetone and blocked in 2% BSA in PBS for 2 hours at RT. Cells were stained with 6G12 hybridoma supernatant at 1:10 at 4C O/N, followed by incubation with anti mouse-Alexa Fluor 488 coupled secondary antibody for 1h at RT. Nuclei were counter-stained with Hoechst 33342.
Simple Western: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Simple Western: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Simple Western: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - Image shows a specific band for Cas9 in 0.2 mg/mL of HeLa Cas9 lysate but not in Hela WT lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system. Observed molecular weight is ~158 kDa.
Chromatin Immunoprecipitation: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) [NBP2-52398] - NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA were fixed with formaldehyde, harvested and sonicated to get 200-500bp DNA fragments. 50ug chromatin was incubated over night at 4C with the indicated antibodies (200ul hybridoma SN, 5ug anti-Flag [M2, Sigma]) followed by incubation with protein G beads for 3h at 4C. After washing chromatin was eluted from the beads and crosslinking was reversed over night at 65C. After a proteinase K digestion step, DNA was separated using phenol/chloroform/isoamyl alcohol, precipitated with ethanol/sodium acetate and dissolved in water. For qPCR, primers either targeting the GFP gene or as negative control non-targeted regions (Ppap2c +7122 and Prkcd +24069 from transcription start) were used.
Western Blot: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Western Blot: CRISPR-Cas9 Antibody (6G12)C-terminusBSA Free [NBP2-52398]

Western Blot: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - Whole cell protein from 293T cells transfected with Cas9-Flag (~150 kDa) 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 ug/mL anti-Cas9 (6G12) in 1% milk, and detected with an anti-mouse HRP secondary antibody using chemiluminescence.
Immunoprecipitation: CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free [NBP2-52398]

Immunoprecipitation: CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free [NBP2-52398]

Immunoprecipitation: CRISPR-Cas9 Antibody (6G12) - C-terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - HEK293 cells expressing Flag-SpCas9 were lysed under native conditions. SpCas9 was immunoprecipitated at 4C from 300 ug of whole cell lysate with the 6G12 antibody and a 1:1 mixture of protein A/G sepharose. After 4x washing, the bound proteins were boiled off the beads, separated by 7.5% SDS-PAGE and transfered to nitrocellulose membranes, and SpCas9 was detected with a rabbit polyclonal Cas9 antibody. After washing, the membranes were incubated with secondary HRP-coupled antibody and bands were visualized by ECL and exposure of X-ray films.

Applications for CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:500

Simple Western

10-20 ug/ml

Western Blot

1:1000

Formulation, Preparation, and Storage

Purification

Protein G 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-associated protein 9 nuclease

Additional CRISPR-Cas9 Products

Product Documents for CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free

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Product Specific Notices for CRISPR-Cas9 Antibody (6G12) - C-terminus - 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.

Citations for CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free

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Protocols

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FAQs for CRISPR-Cas9 Antibody (6G12) - C-terminus - BSA Free

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  • Q: What is the immunogen sequence for your CRISPR-Cas9 antibody NBP2-52398?

    A: CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] is generated against a recombinant C-terminal fragment of Streptococcus pyogene CRISPR/Cas9 [UniProt# Q99ZW2]. Unfortunately, the exact immunogen info is propriatary but we can confirm that the immunogen is outside the catalytic domain of CRISPR-Cas9.

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