Each sample was one well of a 6-well plate, estimated around 300,000 cells per sample. Cells with the indicated treatment were incubated with 10 mg μL−1 of puromycin (Sigma Aldrich) for 30 min. After treatment, cells were washed twice in PBS, suspended in lysis buffer (Cell Signaling), and analyzed by Western blot with an antibody against puromycin (1:5000). α-tubulin (1:3000) was used as a loading control. The amount of newly synthesized protein was quantified using the ProteinSimple Western Blot analysis software and normalized to α-tubulin signal.
Puromycin
Manufactured by Cell Signaling Technology
Sourced in United States
Puromycin is a laboratory reagent used as a selection marker in cell culture experiments. It is an antibiotic that inhibits protein synthesis, allowing for the selective growth of cells that have been genetically modified to express a puromycin resistance gene.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Cell Signaling Technology (2 mentions)
Puromycin, by Merck Group (2 mentions)
Western blot, by Cell Signaling Technology (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Pcmv cat t7 sb100, by Addgene (2 mentions)
Anti sox2 d6d9, by Cell Signaling Technology (2 mentions)
Tracrrna atto 550, by Integrated DNA Technologies (2 mentions)
Custom crrnas, by Integrated DNA Technologies (2 mentions)
Silentfect lipid reagent for rnai, by Bio-Rad (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Psuper retro puro, by Oligoengine (1 mentions)
Luciferase assay system kit, by Promega (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Immobilon western chemiluminescent horseradish peroxidase hrp substrate, by Merck Group (1 mentions)
Lgk974, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
6 protocols using puromycin
1
Protein Synthesis Quantification by Puromycin Assay
2
Ku70 Knockdown Impacts HIV Infection
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For the stable knock down of Ku70 two pSuper.retro.puro (Oligoengine) vectors expressing shRNAs targeting two sites in Ku70 were used. Short duplexes coding for shRNA352 (Sen_shKu70_352/Anti_shKu70_352), shRNA1025 (Sen_shKu70_1025/Anti_shKu70_1025) or shRNACntrl (Sen_shCNTRL/Anti_shCNTRL) were cloned between BglII and HindIII sites. 4 × 105 HEK293T cells were co-transfected with 6 µg of each vector targeting Ku70 or with 6 μg of a vector expressing control shRNA, and the transfected cells were selected with 1 µg/mL of puromycin (Cell Signaling Technology). Individual clones were isolated and Ku70 protein expression was tested by Western blot. Two clones with reduced Ku70 protein expression were selected for further use: clone 8 with a 50% and clone 3 with a 75% reduction in Ku70 expression.
2 × 105 cells of clone 8, clone 3 or control clone were transduced with 1 MOI of HIV-1 viral vector. 24 h or 48 h post infection cells were harvested, cell number was counted and luciferase activity in cell lysates was assayed using the Luciferase assay system kit (Promega).
2 × 105 cells of clone 8, clone 3 or control clone were transduced with 1 MOI of HIV-1 viral vector. 24 h or 48 h post infection cells were harvested, cell number was counted and luciferase activity in cell lysates was assayed using the Luciferase assay system kit (Promega).
3
Protein Synthesis Quantification by Puromycin Assay
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Each sample was one well of a 6-well plate, estimated around 300,000 cells per sample. Cells with the indicated treatment were incubated with 10 mg μL−1 of puromycin (Sigma Aldrich) for 30 min. After treatment, cells were washed twice in PBS, suspended in lysis buffer (Cell Signaling), and analyzed by Western blot with an antibody against puromycin (1:5000). α-tubulin (1:3000) was used as a loading control. The amount of newly synthesized protein was quantified using the ProteinSimple Western Blot analysis software and normalized to α-tubulin signal.
4
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
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To generate CRISPR/Cas9-mediated SOX2KO cell lines, parental CWR-R1 cells were co-transfected with pT2-EF1a-Cas9-P2A-puro and pCMV(CAT)T7-SB100 (#34879, Addgene; Watertown, MA) using Lipofectamine 2000 (Invitrogen) to introduce Cas9 expression. Stable constitutive Cas9 expression was accomplished by SB100 transposase integration of EF1a-Cas9-P2A-puro. Cas9-expressing cells were selected for and maintained with puromycin (1 mg/mL, Invitrogen) 48 h after transfection. Constitutive Cas9 expression was confirmed by western blot (# 14697, Cell Signaling Technologies) after 1 week of puromycin selection. Two custom crRNAs (Integrated DNA Technologies; Coralville, IA) targeting the N-terminus of SOX2 were selected using CHOPCHOP software (https://chopchop.cbu.uib.no ) (SOX2 crRNA #1: 5’-CGGGCCCGCAGCAAACTTCG-3’, SOX2 crRNA #2: 5’-CGCCCGCATGTACAACATGA-3’) and were individually complexed with tracrRNA-ATTO 550 (#1075927, Integrated DNA Technologies) at a 1:1 ratio immediately before transfection. A final concentration of 10 nM crRNA:tracrRNA duplexes were transfected into CWRR1-Cas9 cells using siLentFect Lipid Reagent for RNAi (#1703360, BioRad) following manufacturer guidelines. Limited dilution was performed to isolate three clonal knockout cell lines, and successful knockout of SOX2 was validated by western blot (anti-SOX2(D6D9), #3579, Cell Signaling Technologies).
5
Protein Extraction and Western Blotting Analysis
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Cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) and supplemented with protease and phosphatase inhibitors (Thermo Fischer Scientific, Waltham, MA, USA). The extracted proteins were quantified using a Pierce BCA Protein Assay kit (Thermo Fischer Scientific, Waltham, MA, USA). Twenty micrograms of total protein were then subjected to SDS-PAGE on 10%–12%% gels and was then transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Milwaukee, WI, USA). Membranes were blocked in TBST (Tris-buffered saline, 10 mM Tris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Tween 20) plus 5% nonfat dry milk for 1 h, and the membrane was then probed with the indicated primary and secondary antibodies. Following washes with TBST, membranes were developed using Immobilon Western Chemiluminescent Horseradish peroxidase (HRP) Substrate (Millipore, Billerica, MA, USA). The antibodies used in this study are listed in Table S2 . Other chemicals were obtained from the following suppliers: Sigma (puromycin), Cell Signaling Technology (EGF) and Selleckchem (Houston, TX, USA) (Y-27632 2HCl and LGK-974), and Thermo Fisher Scientific (Lipofectamine 3000).
6
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
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