Performed as in(25 ), but 1 × 105 cells were plated and treated as indicated and pulsed with 50 μM O-propargyl puromycin (OPP, Jena Biosciences) before being collected and washed two times with PBS. Alexa-488 azide (Thermo A10266) was used for conjugation to C-terminally labeled proteins and samples ran on Attune NxT cytometer (Thermo) and data analyzed using FlowJo v9.9.6 (FlowJo).
O propargyl puromycin
Manufactured by Jena Biosciences
Sourced in United States
O-propargyl-puromycin is a chemical compound used in molecular biology applications. It functions as a protein synthesis inhibitor, halting translation by interfering with the ribosome.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Axio observer d1 microscope, by Zeiss (2 mentions)
Alexa fluor click it kit, by Thermo Fisher Scientific (2 mentions)
O propargyl puromycin, by Thermo Fisher Scientific (2 mentions)
Keratin 1 antibody af 87, by Fortrea (2 mentions)
Diva software, by BD (2 mentions)
Facsuite software, by BD (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Actinomycin, by Cambridge Isotopes (2 mentions)
Dulbecco s modified eagle medium dmem media, by Fujifilm (2 mentions)
Alexa488 azide, by Thermo Fisher Scientific (1 mentions)
Attune nxt cytometer, by Thermo Fisher Scientific (1 mentions)
Click it cell reaction buffer, by Thermo Fisher Scientific (1 mentions)
Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Accuri, by BD (1 mentions)
Click it plus edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Click it chemistry reaction, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Alexa 647 azide, by Thermo Fisher Scientific (1 mentions)
12 protocols using o propargyl puromycin
1
Quantifying Protein Synthesis Using OPP
2
Measuring Cell Proliferation in Keratinocytes
3
Measuring Cell Proliferation in Keratinocytes
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48 hours after seeding at clonal density, NFSK were cultured with 50μM O-propargyl-puromycin (Jena Bioscience, NU-931-05) in keratinocyte media for 1 hour. Plates were then fixed with 4% paraformaldehyde for 10 minutes on ice. Colonies were stained following the same protocol described above for immnostaining with EdU using a 1:500 dilution of a Keratin 1 antibody (AF-87, Covance) and an Alexa Fluor Click-IT kit (Invitrogen, C10337) to detect O-propargyl-puromycin. Colonies containing 6 to 10 cells were imaged using a Zeiss Axio-Observer D1 microscope with Zeiss Axiovision software and mean colony fluorescence quantified using NIH ImageJ software.
4
OP-Puro Labeling of Newly Synthesized Proteins
5
Measuring Protein Synthesis with O-Propargyl-Puromycin
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Cells were incubated with 25 μM O‐propargyl‐puromycin (Jena Bioscience) for indicated time at 43°C and fixed using 4% PFA in PBS. As described in Liu et al (Liu et al, 2012 ), the cells were then washed with TBS, permeabilized with TBST (TBS with 0.2% Triton X‐100), and washed with TBS again. CuAAC detection of the incorporated O‐propargyl‐puromycin was performed as previously described (Liu et al, 2012 ). Afterward, cells were washed in TBS and processed for immunofluorescence staining as described above.
6
Simultaneous RNA-Protein Labeling Assay
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EU (BaseClick) and O-propargyl-puromycin (Jena Bioscience) were from the same kit using 5-FAM (Lumiprobe) as a fluorophore. Seventy-two hours after transfection, labeling was performed by adding EU (1 mM) for 15 min and OP-Puro (50 mM) for 15 to 45 min. Labeling was analyzed by flow cytometry (C6, BD Accuri apparatus).
7
Measuring Protein Synthesis in Cells
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For L-homopropargylglycine incorporation assay, 4 × 106 BM cells or transduced TF-1 cells were incubated for 40 min at 37°C in 24-well plates in 1 ml RPMI 1640 without methionine (Gibco) supplemented with 10% FBS (Gibco). L-homopropargylglycine (Jena Bioscience; 50 µM final concentration) was added to the culture medium for 1 h 50 min, and then cells were collected and washed with 1% BSA in PBS. Cells were stained for cell-surface antigens as described earlier. The subsequent fixation/permeabilization and azide-alkyne cycloaddition steps were performed using the Click-iT Plus EdU Flow Cytometry Assay Kit (Life Technologies). Cells were resuspended in 3% FBS in PBS and analyzed by flow cytometry.
For in vivo protein synthesis measurement, mice were injected intraperitoneally with 1 mg O-propargyl-puromycin (Jena Bioscience), and BM cells were harvested 1 h later. The subsequent staining, fixation/permeabilization, and azide-alkyne cycloaddition steps were the same as described for L-homopropargylglycine.
For in vivo protein synthesis measurement, mice were injected intraperitoneally with 1 mg O-propargyl-puromycin (Jena Bioscience), and BM cells were harvested 1 h later. The subsequent staining, fixation/permeabilization, and azide-alkyne cycloaddition steps were the same as described for L-homopropargylglycine.
8
Quantifying Cellular Protein Synthesis
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CTLs that had been seeded as described in “24-hour hypoxia treatments” were counted 24 hours later and resuspended to a final concentration of 1x106 cells/ml in fresh RPMI 1640 with 10% FBS, 50 units/ml penicillin-G, 50 μg/ml streptomycin, 50 μM β-mercaptoethanol and 20 ng/ml IL-2 equilibrated to the appropriate oxygen atmosphere. The CTLs were incubated with 20 µM O-propargyl-puromycin (OPP, Jena Bioscience) for 10 minutes at 37°C in normoxia (18% O2) or hypoxia (1% O2). Control cells cultured at 18% O2 were pre-treated with cycloheximide for 30 minutes before addition of OPP. OPP incorporation into polypeptide chains was terminated by fixing cells with 1% paraformaldehyde for 15 minutes at 22°C. After fixation, cells were washed in 0.5% (v/v) FBS in PBS before being permeabilised with 0.5% (v/v) Triton X-100 in PBS for 15 minutes at 22°C. The incorporated OPP was then labelled with Alexa 647-azide using a standard Click-IT chemistry reaction (ThermoFisher Scientific) for 30 minutes at 22°C. Cells were washed twice with 0.5% FBS (v/v) in PBS, before being resuspended in 0.5% FBS (v/v) in PBS for analysis. Data were acquired on a LSRFortessa flow cytometer with DIVA software (BD Biosciences). Data analysis was performed with FlowJo software (Treestar).
9
Measuring Protein Synthesis Dynamics
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Cells were treated with O-propargyl-puromycin (OPP, Jena Bioscience) for 10 min, and the incorporation of the aminoacyl-tRNA mimetic into newly synthesized polypeptides was measured by labeling the OPP was with Alexa 647-azide (Invitrogen) using a standard Click-IT chemistry reaction (Invitrogen). Cells were analyzed using a FACSVerse flow cytometer with FACSuite software (BD Biosciences) and analyzed with FlowJo software (Treestar). The protein mass of cells following different treatments was determined by BCA assay as per manufacturer’s instructions (Pierce).
10
SILAC-Based Protein Synthesis Profiling
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HeLa cells obtained from ATCC were cultured in Dulbecco's Modified Eagle Medium (DMEM) media (Fujifilm Wako, Osaka, Japan) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, USA) and 100 μg/mL kanamycin. For pulse labeling, the cell culture medium was switched to arginine-and lysine-free DMEM (Thermo Fisher Scientific, Waltham, USA) supplemented with 30 μM O-propargyl-puromycin (OPP) (Jena Bioscience, Jena, Germany), 10% FBS and either "heavy" amino acids [0.398 mM L-( 13 C6, 15 N4)-arginine (Arg"10") and 0.798 mM L-( 13 C6, 15 N2)-lysine (Lys"8")] or "medium" amino acids [0.398 mM L-( 15 N4)-arginine (Arg"4") and 0.798 mM L-(D4)-lysine (Lys"4")] (Cambridge Isotope Laboratories, Tewksbury, USA), and incubated for 2 h. For actinomycin D (actD) treatment, HeLa cells were first pre-incubated with 50 nM actD for 2 h, and subsequent pulse labeling with OPP and SILAC amino acids was performed in the presence of 50 nM actD as described above. Of note, the effect of "light" amino acids (e.g., derived from FBS or by recycling) on SILAC labeling was negligible (Supplementary Fig. 1A), consistent with our previous report (12) . All cells were maintained in a humidified 37°C incubator with 5% CO2.
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