Protein synthesis rate was measured using the Click-iT Plus OPP Protein Synthesis Assay (C10456, Thermo Fisher) according to manufacturer’s protocol. Geometric mean fluorescence intensity (normalized to vehicle) was used as an indicator of the relative translation rate.
Click it plus opp protein synthesis assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT Plus OPP Protein Synthesis Assay is a tool used to detect and quantify newly synthesized proteins in cell cultures. It utilizes a methionine analog, OPP (O-Propargyl-Puromycin), which is incorporated into nascent polypeptides during translation. The incorporated OPP can then be detected using a fluorescent dye, enabling the measurement of protein synthesis rates.
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Lab products found in correlation
40 m cell strainer, by BD (1 mentions)
Sorp lsr 2 analytic flow cytometer, by BD (1 mentions)
Biotracker cystine fitc live cell dye, by Merck Group (1 mentions)
Harmony 3, by PerkinElmer (1 mentions)
Operetta hts imaging system, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
7 protocols using click it plus opp protein synthesis assay
1
Measuring Protein Synthesis Rates
2
Protein Synthesis Measurement in Cell Lines
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Protein synthesis rate in hi12, hFF, and RKO was measured using Click-iT® Plus OPP Protein Synthesis Assay (Thermo Fischer Scientific). Briefly hi12, hFF, and RKO were treated with OPPuro at exponential growth phase and samples were processed according to manufacturer’s protocol and prepared for flow cytometry analysis. Single-cell suspension was prepared by filtering through 40 µm cell strainers (BD Falcon). NuclearMask™ Blue stain was used to gate on cells, after which mean fluorescence intensity (MFI) of the FITC channel was calculated. A SORP BD LSRII Analytic Flow Cytometer (BD Biosciences) was used for acquisition and the data was analyzed with Flowjo 8.8.6 (Tree Star Inc., OP, USA). P-values were calculated using a two-sided Student t-test.
3
Dexa and Obestatin Effects on Myotube Protein Synthesis
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Protein synthesis was measured in C2C12 and KM155C25 myotubes using Click‐iT®Plus OPP Protein Synthesis Assay (Thermo Fisher Scientific, Life Technology, Rockford, IL, USA). Murine or human myotubes were treated with Dexa (1 μM) in the presence or absence of obestatin (10 nM), or with obestatin (10 nM) in the presence or absence the protein synthesis inhibitor, cycloheximide (100 μM) for 24 h. Myotubes were then incubated with 20 μM Click‐iT OPP in cell culture medium for 30 min at 37°C. The cells were washed with PBS and fixed with ethanol and permeabilized by 0.5% Triton X‐100. Click‐iT reaction was carried out according to manufacturer's instructions. HCS NuclearMask™Blue Stain was used to nuclei counterstain. Protein synthesis was measured as the mean intensity of the fluorescent signal and normalized to cell number assessed by the blue NuclearMask signal.
4
Quantifying Protein Synthesis and Amino Acid Uptake
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Protein synthesis rate was measured using the Click-iT Plus OPP Protein Synthesis Assay (Thermo Fisher, C10456) according to manufacturer’s protocol. Geometric mean fluorescence intensity (compared to vehicle) was used as an indicator of the relative translation rate. AA uptake in autophagy-deficient HSCs was measured by the flow cytometry-based kynurenine (Kyn) assay as previously described [27 (link)], and cystine uptake was assessed using BioTracker Cystine-FITC Live Cell Dye (Sigma-Aldrich, SCT047).
5
Quantifying Protein Synthesis in Cells
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Protein synthesis was measured using Click- iT® Plus OPP Protein Synthesis Assay (Molecular Probes, Grand Island, NY). Cells on D0 (hESC) or D15 (cardiomyocytes) were seeded in matrigel-coated 96-well plates, and after 48 h, the staining and detection was performed following the manufacturer’s instructions. The quantitative analysis was performed using an Operetta HTS imaging system (PerkinElmer, Waltham MA, USA). Images of 25 fields per well were evaluated with Harmony 3.5.2 software (PerkinElmer). Fluorescence intensities were measured at the cytoplasm regions around the nucleus. For each condition, 1400 cells were randomly chosen for intensity analysis.
6
Immunofluorescent Characterization of Neural Stem Cells
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Expression of Chd5, nestin, vimentin, Map2, Gfap, Mash1, GFP, H3K27me3, H3K27ac, and actin (i.e., β-actin) and in NSCs and P1 cortices was assessed using immunofluorescent microscopy (Vestin & Mills, 2013 (link)). Protein synthesis was assessed using the OP-puro–based protein assay (Click-iT Plus OPP Protein Synthesis Assay; Molecular Probes). The signal intensities of vimentin, nestin, and OP-puro were quantitated using Volocity software. OP-puro–positive, Mash1-positive, Map2-positive, and Gfap-positive cells were quantitated using the Cell Counter Plugin feature of ImageJ software.
7
Quantifying Protein Synthesis by Click-iT Assay
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Protein synthesis rate was monitored by the Click-iT Plus OPP Protein Synthesis Assay (Molecular Probes, Thermo Fisher Scientific). 200,000 cells were incubated with 20 µM Click-iT OPP (O-propargyl-puromycin) in 100 µl of complete medium for 30 min at 37ºC and 5% CO2. Pre-treated cells with 1 µM cycloheximide (CHX, Sigma-Aldrich) for 24 hours were used as a control of translation inhibition. Cells were fixed with 2% paraformaldehyde for 10 min and permeabilized with 0.25% Triton X-100 for 5 min. Staining was performed following manufacturer's protocol in 100 µl of reaction volume.
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