P493-6 cells were labeled for nascent protein synthesis using Click-iT AHA metabolic labeling reagent (C10102; Invitrogen) per the manufacturer’s instructions. Briefly, following doxycycline treatment (0.1 μg/ml), cells were incubated in methionine-free medium for 30 min before AHA labeling for 1 h. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 15 min followed by one wash with 3% BSA. Cells were then stained using Alexa Fluor 488 Alkyne (A10267; Invitrogen) with Click-iT Cell Reaction Buffer Kit (C10269; Invitrogen). Changes in MFI as a measure of newly synthesized protein were detected by Flow cytometric analysis.
Click it aha metabolic labeling reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT AHA Metabolic Labeling Reagent is a chemical labeling tool used to study protein synthesis in cells. It enables the detection and quantification of newly synthesized proteins by providing a means to incorporate a chemical tag into nascent polypeptides.
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Lab products found in correlation
2 protocols using click it aha metabolic labeling reagent
1
Quantifying Nascent Protein Synthesis
2
Measuring Protein Synthesis in PANC-1 Cells
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PANC-1 cells were labeled for protein synthesis using Click-iT® AHA metabolic labeling reagent as per the manufacturer’s instructions (Invitrogen; Thermo Fisher Scientific, Inc.; Waltham, MA, USA). Briefly, cells were treated with either rapamycin (for 30 min, 1 h, 2 h, or 4 h) or DMSO. Cells were incubated in a methionine-free medium for 30 min before AHA labeling for 1 h. Then, they were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 15 min, and then washed with 3% BSA. Cells were stained using Alexa Fluor 488 Alkyne with the Click-iT Cell Reaction Buffer Kit (both Invitrogen; Thermo Fisher Scientific, Inc.; Waltham, MA, USA). Flow cytometry was used to detect changes in mean fluorescence intensity.
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