![Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody Pack [NBP2-52986] - 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 (diluted 1:10) at 4C o/n, followed by incubation with anti mouse-AF488 coupled secondary antibody for 1 h at RT. Nuclei were counter-stained with Hoechst 33342.](http://images.novusbio.com/fullsize/CRISPR-Cas9-Antibody-Pack-Immunocytochemistry-Immunofluorescence-NBP2-52986-img0004.jpg "Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody Pack [NBP2-52986] - 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 (diluted 1:10) at 4C o/n, followed by incubation with anti mouse-AF488 coupled secondary antibody for 1 h at RT. Nuclei were counter-stained with Hoechst 33342.")
| Reactivity | BaSpecies Glossary |
| Applications | WB, Simple Western, ICC/IF, IHC, IP, ChIP |
| Description | This pack contains 1 vial each of: NBP2-36440SS (0.025 mL), NBP2-52398SS (0.025 mL). |
| Immunogen | See individual datasheets. |
| Dilutions |
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| Application Notes | See individual datasheets of components for their validated applications |
| Storage | Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles. |
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Recent advances in CRISPR-Cas9 The CRISPR-Cas9 genome-engineering tool is a powerful opportunity for researchers to study individual gene function. CRISPR-Cas9, abbreviated for Clustered Regularly Interspaced Short Palindromic Repeats, is a bacterial defense system that can be r... Read full blog post. |
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Top 4 Reasons: Why Use CRISPR-Cas9 Antibodies and How? 1. Verification of the success of transfectionWhy- If the CRISPR-Cas9 transfection is not successful, it would not be relevant to relate the observations from transfected cells to the expected outcome of gene editing experiment. How- CRISPR-Cas... Read full blog post. |
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CRISPR/Cas9: Keep your friends close, but your viruses closer "CRISPR", or clustered regularly interspaced short palindromic repeats, is an ancient bacterial mechanism that prevents the invasion of foreign pathogens to a host organism. Specifically, the CRISPR sequence has been identified as a si... Read full blog post. |
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Thomson Reuters Predicts 2016 Nobel Prize Winners Here at Bio-Techne we always look forward to the annual announcements of winners of the highly coveted Nobel Prize – the greatest award in science. How can you go about predicting which scientists might be in line for a life-changing phone-call fro... Read full blog post. |
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Meeting Report: 2nd International Antibody Validation Meeting Bio-Techne brands Novus Biologicals® and R&D Systems® were proud to support the 2nd International Antibody Validation Meeting held at Bath University, on the 15-16 September, 2016. Almost 100 participants from around the world, including funde... Read full blog post. |
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Application Highlight: Recent uses of TERF2 in immunofluorescence (IF) Telomeres are a region of repeat nucleotide sequences located at the end of chromosomes to protect our DNA from becoming damaged via end-to-end fusion. TERF2, or telomeric-repeat binding factor 2, is important for telomere integrity and aids in th... Read full blog post. |
![Western Blot: CRISPR-Cas9 Antibody Pack [NBP2-52986] - Hela cells and Hela cells expressing Flag-tagged S.pyogenes Cas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/ul Doxycyclin and lysed under native conditions. 30ug of whole cell lysate per lane was separated by 7.5% SDS-PAGE, transfered to nitrocellulose membrane and incubated with 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). A 1 min exposure is shown. Note the specific band of CRISPR-Cas9 with a molecular weight of 158.4 kDa.](http://images.novusbio.com/fullsize/CRISPR-Cas9-Antibody-Pack-Western-Blot-NBP2-52986-img0005.jpg "Western Blot: CRISPR-Cas9 Antibody Pack [NBP2-52986] - Hela cells and Hela cells expressing Flag-tagged S.pyogenes Cas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/ul Doxycyclin and lysed under native conditions. 30ug of whole cell lysate per lane was separated by 7.5% SDS-PAGE, transfered to nitrocellulose membrane and incubated with 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). A 1 min exposure is shown. Note the specific band of CRISPR-Cas9 with a molecular weight of 158.4 kDa.")
![Chromatin Immunoprecipitation (ChIP): CRISPR-Cas9 Antibody Pack [NBP2-52986] - ChIP analysis of CRISPR-Cas9 antibody clones 7A9-3A3 and 6G12 on NIH3T3 cells which were stably expressing GFP-H2B, nuclease dead Cas9 (Flag tagged), and a GFP-targeting gRNA. The cells 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 supernatants of clones 7A9 and 6G12; 5ug Flag antibody) 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.](http://images.novusbio.com/fullsize/CRISPR-Cas9-Antibody-Pack-Chromatin-Immunoprecipitation-ChIP-NBP2-52986-img0001.jpg "Chromatin Immunoprecipitation (ChIP): CRISPR-Cas9 Antibody Pack [NBP2-52986] - ChIP analysis of CRISPR-Cas9 antibody clones 7A9-3A3 and 6G12 on NIH3T3 cells which were stably expressing GFP-H2B, nuclease dead Cas9 (Flag tagged), and a GFP-targeting gRNA. The cells 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 supernatants of clones 7A9 and 6G12; 5ug Flag antibody) 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 Pack [NBP2-52986] - WB analysis of Cas9 protein in lysate of CRISPR-Cas9 transfected 293T cells using anti-Cas9 antibody clone 7A9-3A3 at 2ug/ml concentration.](http://images.novusbio.com/fullsize/CRISPR-Cas9-Antibody-Pack-Western-Blot-NBP2-52986-img0003.jpg "Western Blot: CRISPR-Cas9 Antibody Pack [NBP2-52986] - WB analysis of Cas9 protein in lysate of CRISPR-Cas9 transfected 293T cells using anti-Cas9 antibody clone 7A9-3A3 at 2ug/ml concentration.")
![Immunoprecipitation: CRISPR-Cas9 Antibody Pack [NBP2-52986] - 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.](http://images.novusbio.com/fullsize/CRISPR-Cas9-Antibody-Pack-Immunoprecipitation-NBP2-52986-img0002.jpg "Immunoprecipitation: CRISPR-Cas9 Antibody Pack [NBP2-52986] - 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.")
![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.](https://images.novusbio.com/images/CRISPR-Cas9-Antibody-7A9-3A3-N-Terminus-Simple-Western-NBP2-36440-img0013.jpg "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.")