CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus - BSA Free

Simple Western: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Image shows a specific band for Cas9 (observed molecular weight ~158 kDa) in HeLa Cas9 lysate but not in Hela WT lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Hela cells were transiently transfected with an N-terminally Flag-tagged S. pyogenes Cas9 expression vector. The cells were stained with the Cas9 antibody followed by anti mouse-AF488 coupled secondary antibody. Nuclei were counter-stained with Hoechst 33342.

Immunoprecipitation: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - HEK293T expressing N-terminally Flag-tagged S.pyogenes Cas9 were lysed 72h post transfection by resuspending the cells in Hunt buffer and subjecting to 3 freeze-thaw cycles in liquid nitrogen/ice. Proteins were immunoprecipitated from 100ug of whole cell lysate for 1h at 4C with Cas9 supernatant followed by incubation for 1h at 4C with a 1:1 mixture of protein A/G sepharose beads, or for 2h at 4C with Cas9 ab crosslinked to a 1:1 mixture of protein A/G sepharose beads. Beads were washed 2x with Hunt buffer and 1x with TBS. Bound proteins were eluted by boiling in Laemmli, separated by SDS-PAGE and transferred to nitrocellulose. Membrane was blocked, incubated with Cas9 ab, incubated with HRP anti-mouse secondary. *IgG heavy chain

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Analysis of lysate from Cas9 transfected HEK-293T cells using Cas9 antibody clone 7A9-3A3 at 2ug/ml concentration. The signal was developed using HRP-labelled anti-mouse secondary antibody and ECL based detection. Observed molecular weight is ~158 kDa.

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Human HEK293 cells were transfected with the indicated CRISPR-Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. Asterisks indicate that the difference is statistically significant when comparing Cas9-HE, Cas9-HE-Geminin, and Cas9-Geminin to CRISPR-Cas9 in t-test (*P<0.05 or **P<0.005). Relative expression levels of and other fusions were analyzed by western blot with CRISPR-Cas9 and control anti-tubulin antibodies. Protein extracts were obtained with lysis buffer containing 150 mM NaCl. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/s41467-018-03475-7) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - DF-1 stable cell line (chicken fibroblast) expressing Cas9 (left )or wildtype DF-1 cells (right) stained with Cas9 antibody NBP2-36440 and phalloidin and DAPI to visualise F-actin and DNA respectively. Cells fixed with 4% PFA. Antibody at 1:500 overnight at 4C. ICC/IF image submitted by a verified customer review.

Immunohistochemistry-Frozen: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Analysis of a formalin fixed 20um thick frozen section of mouse brain with GBM xenograft tumor areas (GBM cells over expressing SpyCas9 through lentivirus infection). CRISPR-Cas9 antibody (clone 7A9-3A3) was used at 1:50 dilution. The signal was detected using immunofluoresence labeled secondary antibody via Confocal microscopy. Image submitted via verified customer review.

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - 20 ug whole cell lysates from control MEF (MEF-WT) and MEF-Cas9 stable cell line. CRISPR-Cas9 antibody (clone 7A9-3A3) was used at 1:1000 dilution. Observed molecular weight is ~158 kDa. Image submitted via verified customer review.

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Chicken fibroblasts DF-1 cells stably expressing Cas9 in lanes 2 and 4, wild type fibroblasts in lanes 1 and 3 blot, probed with Cas9 antibody. WB image submitted by a verified customer review.

Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - Analysis of Crispr-Cas9 transfected HEK293 cells using CRISPR-Cas9 antibody (clone 7A9-3A3). Red staining represents CRISPR-Cas9 positivity while DAPI stained nuclei are visible in blue color. Image submitted via verified customer review.

Immunoprecipitation: Mouse Monoclonal CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] - LC-MS/MS signals of a surrogate peptide from Cas9 and a Cas9 variant after immunoprecipitation using CRISPR-Cas9 Antibody (7A9-3A3) [Biotin] (NBP2-36440B). Image from a verified customer review.

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus - BSA Free [NBP2-36440] - Identification of “HDR-enhancer” (HE) domain of CtIP. a Schematic diagram of CtIP protein showing different truncated CtIP proteins have been fused to Cas9 & tested for their ability to stimulate HDR. Various sequence features of CtIP, including tetramerization & dimerization domains, & CDK phosphorylation sites S233, T245, & S276, are indicated. b Identification of a domain of CtIP, called HE, spanning aa 1 to 296, which is able to stimulate HDR when fused to Cas9. Human RG37 fibroblasts transfected w/ indicated plasmids expressing Cas9 or Cas9–CtIP derivatives, T2 guide RNA plasmid, & GFP transgene donor w/ homology arms to targeted AAVS1 locus. Expression of fusion proteins examined by WB (Supplementary Fig. 1). Data are from four independent experiments. Error bars indicate standard deviation. c Functional analysis of HE domain. HEK293 cells transfected w/ indicated Cas9 plasmids, T2 guide RNA, & GFP transgene donor w/ homology arms to AAVS1 targeted locus. HDR-mediated transgene integration measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at cleavage site measured by T7E1 assay. Results are expressed as mean of relative HDR or indel frequencies calculated by normalizing every HDR or indel frequency by induced by Cas9, respectively. Asterisks indicate difference is statistically significant when comparing Cas9–CtIP or Cas9–HE derivatives to Cas9 in nonparametric t-test (*P<0.05, **P<0.005, or ***P<0.0005). Data are from four independent experiments. Error bars indicate standard deviation. The relative expression levels of Cas9 & Cas9–HE derivatives analyzed by WB using anti-Cas9 & control anti-tubulin antibodies Image collected & cropped by CiteAb from following publication (//pubmed.ncbi.nlm.nih.gov/29556040), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus - BSA Free [NBP2-36440] - Stimulation of transgene integration by Cas9–HE & Cas9–Geminin. Relative frequencies of HDR & indels induced by Cas9 or fusion of Cas9 to HE domain, Geminin degron, or both. Human HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, & GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequency calculated by normalizing every HDR or indel frequency by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE, Cas9–HE–Geminin, & Cas9–Geminin to Cas9 in t-test (*P<0.05 or **P<0.005). Data are from three independent experiments. Error bars indicate standard deviation. Relative expression levels of Cas9 & other fusions were analyzed by western blot with anti-Cas9 & control anti-tubulin antibodies. Protein extracts were obtained with lysis buffer containing 150 mM NaCl, which resulted in inefficient solubilization of Cas9 fusions with the HE domain compared to those of Cas9 & Cas9–Geminin Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29556040), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

• Reactivity: Ba
• Applications: WB, Simple Western, ICC/IF, IHC, IP, WB, IHC, KO
• Clone: 7A9-3A3
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus - BSA Free Summary

• Immunogen: This CRISPR-Cas9 antibody (7A9-3A3) - N-Terminus was raised against Recombinant Cas9 within the N-terminal region of Streptococcus pyogene. [Uniprot: Q99ZW2].
• Specificity: This CRISPR-Cas9 antibody (7A9-3A3) - N-Terminus is specific toCas9 protein from Streptococcus pyogene serotype M1.
• Isotype: IgG1 Kappa
• Clonality: Monoclonal
• Host: Mouse
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • Immunoblotting reported in scientific literature (PMID 31959836)
  • Immunocytochemistry/ Immunofluorescence 1:500
  • Immunohistochemistry Whole-Mount
  • Immunohistochemistry
  • Immunohistochemistry-Frozen reported in scientific literature (PMID 28153089)
  • Immunoprecipitation
  • Knockout Validated reported in scientific literature (PMID 31959836)
  • Simple Western
  • Western Blot 1:1000
• Application Notes: IF and IHC use of CRISPR-Cas9 antibody (clone 7A9-3A3) on 4% formaldehyde fixed and 20um thick frozen-/cryo-sections reported in scientific literature
• 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.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein G purified

Alternate Names for CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus - BSA Free

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

Background

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

Limitations

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.

Publications for CRISPR-Cas9 Antibody (NBP2-36440)(44)

Publications using NBP2-36440

• Zhang Y, Rabinovsky R, Wei Z et al. Secreted PGK1 and IGFBP2 contribute to the bystander effect of miR-10b gene editing in glioma Molecular Therapy - Nucleic Acids 2023-01-01 (ICC/IF, Bacteria)
• Ellis-Aguilar LD Development of a non-viral delivery nanoplatform for genomic therapy based on iron oxide nanoparticles and recombinant Cas9 for potential use in genetic heterozygous orphan diseases Thesis 2022-01-01 (ICC/IF)
• Nazma F. Ilahibaks, Thomas A. Kluiver, Olivier G. de Jong, Saskia C. A. de Jager, Raymond M. Schiffelers, Pieter Vader, Weng Chuan Peng, Zhiyong Lei, Joost P. G. Sluijter Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes Journal of Extracellular Vesicles 2024-01-08 [PMID: 38191764]
• Li WK, Zhang SQ, Peng WL et al. Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice Nature neuroscience 2023-11-27 [PMID: 38012399]
• Pettitt SJ, Shao N, Zatreanu D et al. A HUWE1 defect causes PARP inhibitor resistance by modulating the BRCA1-?11q splice variant Oncogene 2023-01-01 [PMID: 37491606] (WB)
• Zelceski A, Francica P, Lingg L et al. MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells Cell reports 2023-05-30 [PMID: 37163373]
• Lamb Am, Wang Z, Simmer P et Al. ebony affects pigmentation divergence and cuticular hydrocarbons in Drosophila americana and D. novamexicana bioRxiv 2020-01-01 [PMID: 37035752] (WB)
• Tanaka M, Chuaychob S, Homme M et al. ASPSCR1-TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma Research Square 2021-12-27 [PMID: 37029109] (WB)
• Szczerba M, Johnson B, Acciai F et al. Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1 Science signaling 2023-03-14 [PMID: 36917643]
• Bert Kwanten, Tine Deconick, Christopher Walker, Feng Wang, Yosef Landesman, Dirk Daelemans E3 ubiquitin ligase ASB8 promotes selinexor-induced proteasomal degradation of XPO1. Biomed Pharmacother 2023-01-31 [PMID: 36731340]
• Cappellesso F, Orban MP, Shirgaonkar N et al. Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer Nature cancer 2022-12-01 [PMID: 36522548] (WB, Mouse)
• Abrego J, Sanford-Crane H, Oon C et al. A Cancer Cell-Intrinsic GOT2-PPAR? Axis Suppresses Antitumor Immunity Cancer discovery 2022-10-05 [PMID: 35894778] (WB, Human)
• Sakashita A, Ariura M, Namekawa SH CRISPR-Mediated Activation of Transposable Elements in Embryonic Stem Cells Methods in molecular biology (Clifton, N.J.) 2022-07-07 [PMID: 35796964]
• Unlu G, Prizer B, Erdal R et al. Metabolic-scale gene activation screens identify SLCO2B1 as a heme transporter that enhances cellular iron availability Molecular cell 2022-06-09 [PMID: 35714613]
• Park S, Uchida T, Tilson S et al. A Dual Conditional CRISPR/Cas9 System to Activate Gene Editing and Reduce Off-Target Effects in Human Stem Cells Molecular Therapy - Nucleic Acids 2022-04-01 [PMID: 35615005] (Simple Western)
• Economos NG, Quijano E, Carufe KEW et al. Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation Nucleic acids research 2022-03-03 [PMID: 35235944] (WB)
• Bozoglu T, Lee S, Ziegler T et al. Endothelial Retargeting of AAV9 In Vivo Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2022-01-12 [PMID: 35023328]
• Uchida T, Park SB, Inuzuka T Et al. Genetically edited hepatic cells expressing the NTCP-S267F variant are resistant to hepatitis B virus infection Molecular Therapy - Methods & Clinical Development 2021-12-01 [PMID: 34853804] (WB, Simple Western)
• Bailey ML, Tieu D, Habsid A et al. Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene STAG2 Life science alliance 2021-11-01 [PMID: 34462321] (WB)
• Ah-Fong AMV, Boyd AM, Matson MEH, Judelson HS A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor Molecular plant pathology 2021-03-16 [PMID: 33724663] (WB, Fungi)
• Zhao X, Jin C, Dong T et al. Characterization of Promoters for Adeno-associated Virus Mediated Efficient Cas9 Activation in Adult Cas9 Knock-In Murine Hear Res 2020-01-10 [PMID: 32611519] (WB)
• Alix E, Godlee C, Cerny O et al. The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules Cell Host Microbe 2020-05-27 [PMID: 32526160]
• Liu Y, Chen J, Crisante D et al. Dynamic Cell Programming with Quorum Sensing-Controlled CRISPRi Circuit ACS Synth Biol 2020-06-19 [PMID: 32485106] (WB)
• Yu Y, Leete TC, Born DA et al. Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity Nat Commun 2020-04-28 [PMID: 32345976]
• Kang W, Zhao X, Sun Z et al. Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae Gene Ther. 2020-01-31 [PMID: 32005950] (WB, Bacteria)
• Wu Y, Yang L, Chang T et al. A Small Molecule-Controlled Cas9 Repressible System Mol Ther Nucleic Acids 2020-01-10 [PMID: 32000033] (WB, KO)
• Wilk A, Hayat F, Cunningham R et al. Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity Sci Rep 2020-01-20 [PMID: 31959836] (KO, WB)
• Schick S, Rendeiro AF, Runggatscher K et al. Systematic characterization of BAF mutations provides insights into intracomplex synthetic lethalities in human cancers. Nat Genet. 2019-09-15 [PMID: 31427792] (WB, Bacterial)
• Baumann V Identification and manipulation of chromatin barriers in transcriptional reprogramming Thesis (WB)
• Li X Gap junction protein connexin43 and tunneling nanotubes in human trabecular meshwork cells. Int J Physiol Pathophysiol Pharmacol. 2019-10-15 [PMID: 31777645] (WB, Bacterial)
• Johnston R, Seamon K, Saada E et al. Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection Biosens Bioelectron 2019-06-18 [PMID: 31207570] (ICC/IF, Bacterial)
• Savage AM, Kurusamy S, Chen Y et al. tmem33 is essential for VEGF-mediated endothelial calcium oscillations and angiogenesis Nat Commun 2019-02-13 [PMID: 30760708] (IF/IHC, Bacteria)
• Qiang J, Ma Z, Xie XX et al. Multi-omic analyses reveal minimal impact of the CRISPR-Cas9 nuclease on cultured human cells. J. Proteome Res. 2019-01-23 [PMID: 30672298] (WB, Bacteria)
• Oakes BL, Fellmann C, Rishi H et al. CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification. Cell 2019-01-10 [PMID: 30633905] (WB, Bacteria)
• Melnik S, Dvornikov D, Muller-Decker K et al. Cancer cell specific inhibition of Wnt/beta-catenin signaling by forced intracellular acidification. Cell Discov. 2018-07-03 [PMID: 29977599] (WB, Bacterial)
• Charpentier M, Khedher AHY, Menoret S et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun. 2018-03-19 [PMID: 29556040] (WB)
• Sykora P, Witt KL, Revanna P et al. Next generation high throughput DNA damage detection platform for genotoxic compound screening. Sci Rep. 2018-02-09 [PMID: 29426857] (WB)
• Basila M, Kelley ML, Smith AVB et al. Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS One. 2017-11-27 [PMID: 29176845] (WB, Bacterial)
• Rogers ZN, McFarland CD, Winters IP. A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo. Nat Methods. [PMID: 28530655]
• El Fatimy R, Subramanian S, Uhlmann EJ, Krichevsky AM. Genome Editing Reveals Glioblastoma Addiction to MicroRNA-10b. Mol Ther. 2017-02-01 [PMID: 28153089] (IHC-Fr, WB, ICC/IF)
  
  Details:
  CRISPR-Cas9 antibody (clone 7A9-3A3) was used for WB analysis of lysates from human/mouse primary astrocytes and neurons which were subjected to transductions with miR-10b-editing lentivirus at the MOI levels that led to similar levels of Cas9 expression. WB was also performed on lysates of established orthotopic LN229 glioblastoma/GBM tumors which were subjected to intratumoral Injections of 105 TU of Lentiviral miR-10b-Editing Vectors. The antibody was also used in ICC/IF assay to determine the lentivirus functional titer (through serial dilutions) in LN229 cells, and for IHC on cryo/frozen sections of brain tumor which were left uninfected or were infected with sgRNA G1. IHC was performed on intracranial tumors which were fixed using 4% formaldehydeand were cryo-sectioned to a 20-um-thickness before processing for staining.
• Chew WL, Tabebordbar M, Cheng JK et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods. [PMID: 27595405]
• Choi JG, Dang Y, Abraham S et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther. 2016-04-07 [PMID: 27052803] (ICC/IF)
• Chu VT, Weber T, Graf R et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 2016-01-16 [PMID: 26772810] (WB)
  
  Details:
  CRISPR/Cas9 antibody used for WB on lysates from TAT-Cre/LPS treated naive B cells of three Rosa26LSL-Cas9 F1 mice (Figure 3D).
• Chiou SH, Winters IP, Wang J et al. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev 2015-07-15 [PMID: 26178787] (WB)

Reviews for CRISPR-Cas9 Antibody (NBP2-36440) (8)

Average Rating: 4.6

(Based on 8 reviews)

We have 8 reviews tested in 4 species: Avian, Bacteria, Chicken, Other.

reviewed by: Stephen Terry WB Chicken 09/06/2021

Summary

• Application: Western Blot
• Sample Tested: DF-1 fibroblasts
• Species: Chicken
• Lot: J

Comments

• Comments: membrane blocked in 5% milk TBST
  antibody used 1:1000
  antibody incubated for 20h at 4 degrees

reviewed by: Stephen Terry ICC Avian 08/25/2021

Summary

• Application: Immunocytochemistry
• Sample Tested: DF-1 chicken fibroblasts,fixed DF-1 chicken fibroblast cells
• Species: Avian
• Lot: J

Comments

• Comments: cells fixed with 4%PFA, perm with 0.3% tx100 for 5 mins antibody incubated 1:500 overnight at 4 degrees ***Bio-Techne Response: This review was submitted through the legacy Novus Innovators Program, reflecting a new species or application tested on a primary antibody.***

reviewed by: Verified Customer WB Bacteria 03/20/2017

Summary

• Application: Western Blot
• Sample Tested: MEF (MEF-WT) and MEF-Cas9 stable cell line
• Species: Bacteria

Comments

• Comments: Sample: Whole cell lysates from our control MEF (MEF-WT) and MEF-Cas9 stable cell lines
  
  Loading amount: 20ug
  
  Primary antibody dilution: 1:1,000
  
  Detection: ECL method
  
  Comments on Performance: Good specificity and sensitivity – attached image for a whole membrane

reviewed by: Verified Customer WB Bacteria 03/16/2017

Summary

• Application: Western Blot
• Sample Tested: CRISPR-Cas9 expressing Ewing Sarcoma cell lines (SKES1 and TC71)
• Species: Bacteria

Comments

• Comments: Human Cell Lines tested: Ewing Sarcoma cell lines (SKES1 and TC71)
  
  Application tested: Western Blot
  
  Dilution tested: 1:1000
  
  Detection: ECL method

reviewed by: Verified Customer IHC-Fr Bacteria 03/02/2017

Summary

• Application: Immunohistochemistry-Frozen
• Sample Tested: Xenograft tissue
• Species: Bacteria

Comments

• Comments: Testing: IHC-Frozen
  
  Tissues:
  
  1.LN229-formed glioblastoma xenografts, uninfected or infected with sgRNA G1.
  
  2.GBM8 formed glioblastoma xenograft, treated or not with the mutated nickase version of the virus-encoded Cas9n D10A enzyme and guided by the pair of G1 and G3 sgRNAs.
  
  Fixative: 4% formaldehyde
  
  Section thickness: 20um
  
  Antibody Dilution: 1-50
  
  Detection: Immunofluoresence, Confocal microscopy
  
  Data Images: El Fatimy et al. 2017. Mol Ther. 2017 Feb 1;25(2):368-378 [PMID 28153089]
  
  Performance; Antibody is more specific than other Cas9 antibodies tested.

reviewed by: Verified Customer ICC Bacteria 03/02/2017

Summary

• Application: Immunocytochemistry
• Sample Tested: LN229 cells (human glioblastoma cell line)
• Species: Bacteria

Comments

• Comments: Antibody used at 1:100 dilution in immunocytochemistry with Immunofluorescence based detection to determine the lentivirus functional titer (through serial dilutions) in LN229 cells.

reviewed by: Verified Customer WB Bacteria 03/02/2017

Summary

• Application: Western Blot
• Sample Tested: Human or mouse primary astrocytes and neurons transfected with bacterial Crispr-Cas9,Human primary astrocytes transfected with Crispr-Cas9,Human primary astrocytes,LN229 cells (human glioblastoma cell line) transfected with bacterial Crispr-Cas9,LN229 cells (human glioblastoma cell line),LN229 human glioblastoma cells transfected with Crispr-Cas9,Mouse primary astrocytes transfected with Crispr-Cas9,Mouse primary astrocytes,Primary mouse astrocytes transfected with Crispr-Cas9
• Species: Bacteria

Comments

• Comments: Lysates used:
  1.Human or mouse primary astrocytes and neurons transduced with miR-10b-editing lentivirus at the MOI levels that led to similar levels of Cas9 expression.
  2.Established orthotopic LN229 glioblastoma/GBM tumors that were subjected to intratumoral Injections of 105 TU of Lentiviral miR-10b-Editing Vectors.
  
  Dilutionused: 1-2000
  
  Detection Method: ECL

reviewed by: Verified Customer IF Other 07/27/2016

Summary

• Application: Immunofluorescence
• Sample Tested: HEK293 cells transfected with Cas9
• Species: Other

FAQs for CRISPR-Cas9 Antibody (NBP2-36440). (Showing 1 - 2 of 2 FAQs).

1. Does the antibody NBP2-36440 cross react with the inactive form of CAS9 and CAS9 nickase?
   • Unfortunately, we do not have any information regarding the ability of this clone to detect the Cas9 nickase. Nickase activity involves amino acids 10 and 840 of the Cas9 sequence. This particular antibody was raised against the N-terminal region of the Cas9 protein. It is possible that this product may then detect the modification surrounding amino acid 10, but again, we do not have data at this time that proves this interaction.
2. What is the immunogen sequence for your CRISPR-Cas9 antibody NBP2-36440?
   • CRISPR-Cas9 Antibody (7A9-3A3) - N-Terminus [NBP2-36440] is generated against a recombinant N-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.