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Click it protein synthesis assay kit

Manufactured by Thermo Fisher Scientific

The Click-iT protein synthesis assay kit is a tool used to detect and quantify newly synthesized proteins in cells. It utilizes a reactive chemical group to label newly translated proteins, allowing for their visualization and analysis.

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9 protocols using click it protein synthesis assay kit

1

Pancreatic Cell Characterization Protocol

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Pancreas tissue was harvested following transcardiac perfusion with 4% paraformaldehyde and fixed in 4% paraformaldehyde. Pancreatic sections were stained with antibodies against insulin and glucagon, phosphorylated S6 (p-S6; Ser235/236 for IHC and IFC, Cell Signaling). Nascent protein synthesis was visualized using the Click-iT protein synthesis assay kit (C10428, Life Technologies).
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2

Measuring Protein Synthesis in Cells

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Cells were fixed by 2% PFA, permeabilized by methanol, then incubated with primary (1:300) and secondary antibody (1:2000). To measure translation rates in vivo, mice were injected with OP-Puro (Medchem Source, 50 mg/kg, i.p.), BM was collected one hour later and stained for surface markers and OP-Puro using the Click-iT Protein Synthesis Assay Kit (Life Technologies) (Signer et al., 2014 (link)). To measure translation following Runx1 deletion in vitro, purified Kit+ cells were cultured with 5 μM 4-OHT for 48 hrs, and OP-Puro added 20 minutes before harvesting and staining the cells.
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3

Protein Synthesis Analysis in Oocytes

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To examine the total protein synthesis in ND, HFD, and IGF2-treated HFD oocytes, the Click-iT protein synthesis assay kit (C10428, Life Technologies) was used following the manufacturer’s instructions as described previously [28 ]. Briefly, the MII-stage oocytes from different groups were exposed to 50 μM HPG for the period of 1 hr. at 37 °C with 5% CO2. After treatment, 3.7% paraformaldehyde was used for oocytes fixation and permeabilization was performed with Triton X-100 at room temperature for 20 min. HPG signal intensity is indicative of the total protein synthesis index in oocytes.
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4

Measuring Protein Synthesis in Oocytes

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The protein synthesis assay was performed as described previously [33 (link)] using the Click-iT protein synthesis assay kit (C10428, Life Technologies) following the manufacturer’s instructions. Briefly, the MII-stage oocytes were incubated in culture medium supplemented with 50 μM HPG at 37° C with 5% CO2 for 1 h. Oocytes were fixed with 3.7% formaldehyde followed by permeabilization with 0.5% Triton X-100 for 20 min at room temperature. The HPG signal is indicative of the overall level of translation in oocytes.
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5

Quantifying Nascent Protein Synthesis

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Detection of nascent protein synthesis was assessed with Click-IT protein synthesis assay kit (#C10428, ThermoFisher) and performed following the manufacturer’s protocol. The methionine analog L-homopropargylglycine was added (50 µM) to cultures 30 min before the first sampling of each time point. The detection of click reaction was obtained by incubating fixed cells for 1 h with Alexa Fluor 488 azide in Click-iT cell reaction buffer solution. Fluorescence was recorded using a Leica TSC SP8 confocal microscope and ImageJ. Nascent protein synthesis was assessed by determining the signal intensity and finally normalized by the control condition of each biological replicate. Microscope settings (image exposure times and thresholds) were kept the same during the image acquisition of all the different conditions.
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6

Visualizing Protein Synthesis in Fungi

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To observe translation in fungal cells, a Click-iT protein synthesis assay kit (ThermoFisher) was used per the manufacturer’s instructions. Details are provided in Text S1 in the supplemental material. Cells were imaged by DIC microscopy and by the use of the enhanced green fluorescent protein (EGFP) channel on a Zeiss Axio Imager.MI microscope (Carl Zeiss) at the same exposure time.
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7

Quantifying Protein Synthesis in C. albicans

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To observe translation in C. albicans cells, we used a Click-iT Protein Synthesis Assay Kit (Thermofisher) per the manufacturer’s instructions. WT C. albicans was subcultured from overnight cultures in YPD into minimal media and allowed to grow to log phase (OD600 0.4 – 0.8). After this growth, non-control cultures were treated with 25 μM NSC 697923 for 10 minutes. 50 μM HPG Reagent was used. Cells were fixed with 4% Paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100 in PBS. To quantify translation, cells were imaged on a Biotek Lionheart microscope at 40X magnification using a Texas Red channel at the same exposure time for each image. The same cells were also examined by flow cytometry on a BD Fortessa flow cytometer and analyzed using FloJo (BD Biosciences).
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8

Visualizing RNA and Protein Synthesis in Oocytes

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For nascent transcription assays, oocytes were incubated in M16 medium with IBMX supplemented with 1 mM EU (5-ethynyl uridine) for 2 h, fixed in methanol, then a Click-iT RNA Imaging kit (Thermo Fisher Scientific) was used and visualisation was by confocal microscopy. Changes in protein expression were monitored using a Click-iT Protein Synthesis Assay kit (Thermo Fisher Scientific). Oocytes and two-cell embryos were cultured in M16 medium with the methionine analogue HPG at a dilution of 1:500. Newly synthesised proteins incorporating the HPG were visualised by confocal microscopy.
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9

Measuring Nascent Protein Synthesis with Click-iT Assay

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Nascent protein synthesis was detected by the Click-iT protein synthesis assay kit according to the manufacturer's instruction (Thermo Fisher Scientific, catalog no. C10456). Briefly, cells pretreated with 20 μmol/L Click-iT O-propargyl-puromycin (OPP) for 30 minutes were harvested, fixed, and permeabilized. After washing in PBS, cells were stained in Click-iT Plus OPP reaction cocktail, containing Alexa Fluor 488 azide, for 30 minutes in dark, followed by flow cytometry analysis (17 (link)).
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