Click it hpg

Manufactured by Thermo Fisher Scientific

Click-iT® HPG is a methionine analog that can be used to label newly synthesized proteins in live cells. It allows for the detection and quantification of protein synthesis.

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Lab products found in correlation

Methionine free dmem, by Thermo Fisher Scientific (2 mentions) Click it protein reaction buffer, by Thermo Fisher Scientific (2 mentions) Protein g agarose, by Merck Group (2 mentions) Anti iκbα, by Cell Signaling Technology (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Anti c myc, by Cell Signaling Technology (1 mentions) Anti caprin 1, by Proteintech (1 mentions) Anti cyclin d2, by Santa Cruz Biotechnology (1 mentions) Anti cyclin d1, by Santa Cruz Biotechnology (1 mentions) Ethylene diamine tetraacetic acid (edta), by Corning (1 mentions) Nuclearmask blue stain, by Thermo Fisher Scientific (1 mentions)

4 protocols using click it hpg

Analysis of de novo protein synthesis

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This assay was performed as described^{18 (link), 20 (link)}. Briefly, ST cells were washed with PBS and cultured with methionine-free DMEM (GIBCO-Life Technologies) at 37 °C for 1 h prior to the addition of 1 μM tylophorine and 50 μM Click-iT^® HPG (L-homopropargylglycine [a reactive methionine analog], Invitrogen) for another 1 h and followed by infection of TGEV at an MOI of 7 for 1 or 3 h. The cells were then harvested and subjected to the ‘click’ reaction with 20 µM TAMRA (tetramethylrhodamine, Invitrogen) for labelling the de novo synthesized proteins in Click-iT® Protein Reaction Buffer according to the manufacturer’s protocol (Invitrogen). The resultant cell lysates were immunoprecipitated with anti-IκBα (Cell Signaling Technology) overnight at 4 °C with constant agitation prior to incubation with protein G agarose (Millipore) at 4 °C for another 2 h. After three to five washes, the specific immunoprecipitated protein was eluted and analyzed by western immunoblot analysis with the antibody against TAMRA (Thermo Fisher Scientific).

Yang C.W., Lee Y.Z., Hsu H.Y., Shih C., Chao Y.S., Chang H.Y, & Lee S.J. (2017). Targeting Coronaviral Replication and Cellular JAK2 Mediated Dominant NF-κB Activation for Comprehensive and Ultimate Inhibition of Coronaviral Activity. Scientific Reports, 7, 4105.

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Protein Translation Assay Protocol

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The protein translation assay was adapted from ref. ^{50 (link)}. First, single-cell suspensions were prepared as described above (‘Flow cytometry’). Then, the cells were washed and plated at 1 million cells per well in a V-bottom 96-well plate in methionine-free R10 (Gibco, A1451701) supplemented with 10% FBS, 1× non-essential amino acids (Gibco, 11140050), 10 mM HEPES (Gibco, 15630080, 7.2 to 7.5), 2 mM l-glutamine (Gibco, 25030081), 100 U ml⁻¹ penicillin–streptomycin (Gibco, 15140122) and 14.3 μM beta-mercaptoethanol. The cells were rested at 37 °C for 3 h, then 400 μM Click-iT HPG (Invitrogen, C10186) was added. After 3 h, cells were stained with viability dye and surface antibody cocktail as described above (‘Flow cytometry’). Then, the cells were fixed and permeabilized (BD, 51–2090KZ) for 20 min at room temperature (~22 °C), followed by one wash with perm wash (BD, 51-2091KZ) and one wash with PBS. Next, the Click-iT reaction was performed according to the manufacturer’s protocol (Invitrogen, C10641). Samples were analyzed as described above (‘Flow cytometry’).

Giles J.R., Ngiow S.F., Manne S., Baxter A.E., Khan O., Wang P., Staupe R., Abdel-Hakeem M.S., Huang H., Mathew D., Painter M.M., Wu J.E., Huang Y.J., Goel R.R., Yan P.K., Karakousis G.C., Xu X., Mitchell T.C., Huang A.C, & Wherry E.J. (2022). Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics. Nature immunology, 23(11), 1600-1613.

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Labeling De Novo Protein Synthesis

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HONE-1 cells were washed with PBS and cultured with methionine-free DMEM (GIBCO-Life Technologies) at 37°C for 1 h prior to the addition of 2 μM tylophorine and 25 μM Click-iT® HPG (L-homopropargylglycine, Invitrogen) and incubation at 37°C for 24 h. The cells were then harvested and subjected to the Click Reaction with 20 μM TAMRA (Tetramethylrhodamine, Invitrogen) for labeling the de novo synthesized proteins in Click-iT® Protein Reaction Buffer according to the manufacturers' protocol (Invitrogen). The resultant cell lysates were immunoprecipitated with anti-caprin-1 (ProteinTech Group), anti-c-Myc (Cell Signaling Technology), anti-cyclin D1, anti-cyclin D2 (Santa Cruz Biotechnology), anti-TAMRA (Thermo Scientific) or GAR (Perkin-Elmer) respectively overnight at 4°C with constant agitation prior to incubation with protein G agarose (Millipore) at 4°C for another 2 h. After washes, the specific immunoprecipitated protein was eluted and analyzed by western immunoblot analysis with the antibody indicated.

Qiu Y.Q., Yang C.W., Lee Y.Z., Yang R.B., Lee C.H., Hsu H.Y., Chang C.C, & Lee S.J. (2014). Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget. Oncotarget, 6(4), 2148-2163.

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Measuring Protein Synthesis Inhibition

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To detect CHX-mediated inhibition of protein synthesis, Gli36-mCherry cells in a 24-well plate were prepared in parallel to the EV uptake and EV-RNA translation experiments as described above. At the indicated post-centrifugation time points, culture medium was replaced with methionine- and cysteine-free medium containing Click-iT HPG (Invitrogen) for 30 min, and the cells were washed with PBS, trypsinized with 0.05% trypsin/0.53 mM EDTA (Corning Cellgro), resuspended in PBS, pelleted at 1,500 r.p.m. (maximum rotor radius: 20.78 cm) for 5 min at 4 °C and resuspended in 80% ethanol for 15 min on ice. The fixed cells were conjugated with Alexa Fluor 488 azide to reveal HPG labelling, and stained with NuclearMask Blue stain according to the manufacturer's protocol (Invitrogen). The labelled cells were resuspended in PBS for flow cytometry analysis on a BD LSRII Multi-Laser Analyser (BD Biosciences).

Lai C.P., Kim E.Y., Badr C.E., Weissleder R., Mempel T.R., Tannous B.A, & Breakefield X.O. (2015). Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nature Communications, 6, 7029.

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