Anti-Gpx4 (Abcam, ab41787, 1:2000), Anti-xCT (Abcam, ab37185, 1:2000), Anti-Cas9 (Cell signaling, 14697, 1:1000,), Anti-ß-Actin (Sigma, A1978, 1:10,000), Anti-GCLC (Santa Cruz, sc-166345, 1:1000), Anti-Ascl1 (BD Pharmingen, 556604, 1:1000), Anti-Txnrd1 (Cell signaling, 15140S, 1:1000), Anti-Txnrd2 (Cell signaling, 12029, 1:1000), Anti-Acsl4 (Santa Cruz Biotechnology, sc-271800, 1:2000), Anti-Txn1 (Cell signaling, 2429S, 1:1000), Anti-GAPDH (Cell signaling, 97166S, 1:2000), Anti-FSP1 (previously described55 , kindly provided by M. Conrad, undiluted hybridoma supernatant), Anti-REST1 (ThermoFisher, BS-2590R, 1:1000), Anti-REST1 (Abcam, ab21635, 1:1000), Anti-TXNIP (Cell signaling, 14715S, 1:1000), Anti-CD71 (Santa Cruz, sc-65882, 1:2000), Anti-cMyc (Abcam, ab32072, 1:2000), Anti- NCAM (Invitrogen, PA5-79717, 1:1000), Anti-Vimentin (Abcam, ab137321, 1:1000), Anti-YAP1 (Cell signaling, #4912, 1:1000), Anti-Synatophysin (Invitrogen, MA5-14532, 1:1000), Anti-AGPAT2 (Thermo Fisher, PA5-76010, 1:2000), Anti-AGPAT3 (Thermo Fisher, PA5-101343, 1:2000). HRP-conjugated secondary antibodies: goat-anti-mouse-HRP (Linaris GmBH, 20400-1 mg, 1:10,000), goat-anti-rabbit-HRP (Linaris GmBH, 20402-1 mg, 1:10,000), goat-anti-rat-HRP (Sigma, A9037-1 ml, 1:10,000).
Anti cas9
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Cas9 is a laboratory reagent designed to detect and quantify the presence of Cas9 protein, a key component of the CRISPR gene editing system. It functions by specifically binding to the Cas9 protein, allowing for its identification and measurement in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Anti actin, by Merck Group (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Goat anti rat hrp, by Merck Group (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti yap1, by Cell Signaling Technology (1 mentions)
Anti xct, by Abcam (1 mentions)
Anti txnip, by Cell Signaling Technology (1 mentions)
Anti ascl1, by BD (1 mentions)
Anti cd71, by Santa Cruz Biotechnology (1 mentions)
Anti ncam, by Thermo Fisher Scientific (1 mentions)
Anti acsl4, by Santa Cruz Biotechnology (1 mentions)
Anti gclc, by Santa Cruz Biotechnology (1 mentions)
Anti gpx4, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Tris acetate gradient gel, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
13 protocols using anti cas9
1
Comprehensive Immunoblotting Antibody Panel
2
Western Blot Analysis of CRISPR-Cas9 Proteins
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Protein lysates were made using lysis buffer (20 mM Tris at pH 8.0, 300 mM NaCl, 2% NP40, 20% glycerol, 10 mM EDTA) complemented with protease inhibitors (Roche) and quantified using the BCA protein assay kit (Pierce). Protein lysate was loaded onto a 3%–8% Tris-acetate gradient gel (Invitrogen) and transferred overnight onto PVDF membrane (Millipore) in transfer buffer (238 mM glycine, 80 mM TRIS, 0.01% SDS in water). Membranes were blocked in 5% ELK in PBS-T (0.05% Tween-20), after which they were stained for 4 h at room temperature using the primary antibodies anti-Flag (1:1000; Sigma Aldrich, F1804) and anti-Cas9 (1:1000; Cell Signaling, 14697) in 1% ELK in PBS-T. Pol II (1:400; Santa Cruz Biotechnology, sc-5943) was incubated for 1 h at room temperature in 1% ELK in PBS-T. Membranes were washed three times with 1% ELK in PBS-T and incubated for 1 h with an HRP-conjugated secondary antibody (1:2000; DAKO). Stained membranes were washed three times in 1% ELK in PBS-T and then developed using SuperSignal ECL (Pierce).
3
Western Blot Analysis of DNA Repair Proteins
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Protein samples were separated on Novex 4–12% Bis‐Tris gradient gels (Invitrogen) using MOPS SDS running buffer or on Novex 3–8% Tris‐acetate gels (Invitrogen) using Tris‐acetate SDS running buffer and transferred onto Immobilon‐FL membranes (Merck Millipore). The primary antibodies used to analyse protein expression were as follows: anti‐Ku80 (M‐20 goat polyclonal, Santa Cruz sc‐1485), anti‐Lig4 (rabbit polyclonal, Abcam 80514), anti‐Cas9 (mouse monoclonal, 7A9‐3A3 Cell Signaling), anti‐Tubulin (clone DM1a, Sigma), anti‐polymerase mu (rabbit monoclonal, ab157465, Abcam) and anti‐polymerase lambda (rabbit polyclonal, kind gift of Prof. Luis Blanco). The secondary antibodies CF680 goat anti‐rabbit IgG and CF770 goat anti‐mouse IgG (Biotium) and the Odyssey infrared imaging scanning system (LI‐COR biosciences) were used to detect protein expression.
4
Immunohistochemistry for Ascl1, Chromogranin, and Synaptophysin
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For immunohistochemistry (IHC), tissue samples were cut as 3 µm thick sections from formalin-fixed paraffin embedded (FFPE) tissues. IHC staining was performed using a Bond Max automated staining system (Leica) using the antibodies: anti-Ascl1 (Abcam #211327, 1:400), anti-Chromogranin (Abcam #52983, 1:250), anti-Synaptophysin (Abcam #32127, 1:1000). GLuc and Cas9 staining was performed manually using the antibodies: anti-GLuc (Prolume Ltd, 1:1000), anti-Cas9 (Cell Signaling #19526, 1:400). Images were acquired using the Leica Aperio Versa slide-scanner and Leica Aperio eSlide Manager software v. 1.0.3.37. IHC images were analyzed quantitatively using the Aperio ImageScope software v. 12.3.2.8013. Tumors were marked and outlined by individuals blinded to the experimental setup and their area quantified in ImageScope. Tumor burden was calculated as percentage of tumor area to total lung area.
5
Cas9 Protein Expression Analysis
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Cell pellets were lysed and protein concentrations were quantified by BCA assay (ThermoFisher). Western blots were performed on protein lysates (60 μg/well). Primary antibodies used were anti-Cas9 (Cell Signaling Technology, 14697) and anti-H3 (Cell Signaling Technology, 14269). Protein was visualized using an IRDye800CW-conjugated secondary antibody (Licor, 926–32210).
6
Immunofluorescence Imaging of Cas9 and DNA Repair Proteins
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U2OS cells were reverse transfected using PEI (Sigma-Aldrich). 50,000 cells were seeded per well of μ-Slide 8-well (Ibidi) chambered coverslip plates. Pre-extraction was performed using 0.1% Tween in PBS 24 h after reverse transfection. Cells were then fixed with 4% para-formaldehyde and fixed cells were processed for immunofluorescence using the following antibodies: anti-Cas9 (Cell Signalling, 14697), anti-TRF1 (Abcam, ab1423), anti-MLH1 (ThermoFisher, A300-015A) and anti-GFP (Abcam, ab6556). Primary antibodies were diluted 1:500 and secondary antibodies (Alexa Fluor® 568 goat anti-mouse and Alexa Fluor® 488 goat anti-rabbit, LifeTechnologies) were diluted 1:2000.
7
Quantitative Protein Analysis of Cas9
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Cells were lysed and protein was extracted from cultured cells using RIPA buffer (Thermo Fisher) and the protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher). SDS-PAGE system (Biorad) was next used to electrophorese approximately 20 µg of protein per sample. Separated proteins were transferred to solid-phase membrane supports using the BIO-RAD blotting system and the primary antibodies (anti-Cas9 from Cell Signaling and anti-GAPDH from ProteinTech, Rosemont, IL, USA) were used to detect protein expression levels.
8
Protein Analysis of Cell Lysates
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Whole-cell lysates were prepared using cell extraction buffer (FNN0011, Invitrogen) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, #1861282) and Basemuncher (BM0025, Expedeon). Protein extracts were prepared at 0.2 μg/μL, loaded, separated, and detected by the Sally Sue Simple Western system (ProteinSimple). The antibodies used in this study were used on Sally Sue at the following dilutions: anti-SMAD4 1:200 (#ab40759, Abcam), anti-GAPDH 1:200 (#2118, Cell Signaling Technology), anti-Cas9 1:200 (#14697, Cell Signaling Technology), anti-RAB10 1:100 (#8127, Cell Signaling Technology), anti SMAD3 1:100 (#9523), anti SMAD2 1:100 (#5339), and anti actin 1:100 (#4967).
9
Cas9 Protein Detection by Western Blot
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The western blotting procedure has been previously described.[24 ] The cell protein elution solution was prepared using lysis buffer with a protease inhibitor (Nacalai Tesque, Inc.) and a phosphatase inhibitor (Nacalai Tesque, Inc.). We used the following antibodies for immunodetection: anti‐Cas9 (#14697; Cell Signaling Technology) and anti‐β‐actin (A5316; Sigma–Aldrich).
10
Protein Extraction and Western Blot Analysis
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Cell pellets were harvested and resuspended in Mammalian Cell & Tissue Extraction Kit buffer (BioVision Incorporated, K269) and incubated for 10 min on ice. Protein lysates were collected after centrifugation at 14,000 × g for 20 min at 4 °C. The total amount of protein was quantified using the Qubit Protein Assay Kit and a Qubit Fluorometer (Thermo Fisher). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were performed using pre-cast NuPAGE Bis-Tris 4–12% mini-gels (Invitrogen) with 1X MOPS buffer (Invitrogen), following the manufacturer’s instructions. The primary antibodies anti-ILF2/NF45 (Santa Cruz, sc365068, dilution 1:500), anti-vinculin (Sigma, V9131, dilution 1:2000), anti-γH2AX (Cell Signaling, 2577 S, dilution 1:500), anti-cleaved caspase 3 (Cell Signaling, 9661 S, dilution 1:500), and anti-Cas9 (Cell Signaling, 14697 S, dilution 1:1000), in addition to secondary anti-mouse and anti-rabbit digital antibodies (Kindle Biosciences LLP, dilution 1:2000), were used. Membranes were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and imaged using a KwikQuant Imager and software (Kindle Biosciences LLP).
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