This was done as previously described (44 (link)). Briefly, newly synthesized proteins were labeled using the Click-iT Protein Labeling kit (Invitrogen). Isogenic Nalm-6 cells were cultured in fresh medium for 24 hrs, washed twice with PBS, and resuspended in methionine-free RPMI 1640 medium (Gibco) supplemented with 10% FCS for 30 min, at which point the methionine analog L-azidohomoalanine (AHA; Invitrogen) was added (50 μM) to allow incorporation of AHA into nascent proteins. MG-132 (Sigma, M7449; 5 μM) was added at 4 hrs before the methionine replacement. Protein was extracted from the cells and 150 μg of total protein was used in the cross-linking of AHA-labeled nascent proteins to alkyne-derivatized biotin in Click-iT Protein Reaction Buffer (Invitrogen) according to the manufacturer’s instructions. Biotin-cross-linked nascent protein was captured overnight with streptavidin-coated Dynabeads (M-280, Invitrogen) and eluted. The whole volume of AHA-labeled, biotin-cross-linked, streptavidin-precipitated protein was separated by SDS–PAGE.
Click it protein labeling kit
Manufactured by Thermo Fisher Scientific
The Click-iT Protein Labeling kit is a tool used to label and detect newly synthesized proteins in cells. It utilizes a chemical reaction to incorporate a modified amino acid into the protein of interest, enabling its visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Click it protein reaction buffer, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
L azidohomoalanine aha, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Dynabeads m 280, by Thermo Fisher Scientific (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Methionine free rpmi, by Thermo Fisher Scientific (1 mentions)
Proteosilver, by Merck Group (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
4 protocols using click it protein labeling kit
1
Metabolic Labeling of Nascent Proteins
2
Labeling and Capturing Nascent Proteins
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Newly synthesized proteins were labeled using the Click-iT Protein Labeling Kit (Invitrogen) as previously described (34 (link)). 30 × 106 OCI-Ly1 cells were pretreated with either vehicle or selinexor (1 μmol/L) for 2 hours. After one wash, cells were resuspended in methionine-free RPMI1640 medium (Gibco) supplemented with the methionine analog L-azidohomoalanine (AHA; 50 μmol/L). Each flask was then divided into two flasks (15 × 106 cells each) and cells were exposed to vehicle, etoposide (3 μmol/L), selinexor (1 μmol/L), or the combination of selinexor and etoposide for 6 hours. Cells were then harvested and resuspended in lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 1% SDS) containing a fresh protease inhibitor cocktail. 150 μg of protein was used to crosslink the AHA-labeled nascent proteins to alkyne-derivatized biotin in the Click-iT Protein Reaction Buffer (Invitrogen) according to the manufacturer's instructions. The resulting protein pellet was resolubilized in 1% SDS, followed by quenching of the SDS with 6% NP-40, both supplemented with a protease inhibitor cocktail. Biotin-crosslinked nascent proteins were then captured overnight with streptavidin-coated Dynabeads M-280 (Invitrogen) and then eluted from the beads by boiling the samples for 5 min in 2% SDS loading buffer for Western blot analysis.
3
Metabolic Labeling of Murine B Cells
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For murine primary B cell experiments, cells were isolated and either rested or stimulated for 16–20 hr with 10 μg/mL anti-IgM. For BJABs, cells incubated either in media or with 100 ng/mL doxycycline for 48 hr were centrifuged over a ficoll gradient (GE Healthcare Lifesciences, Marlborough MA) to generate a pure live cell population. Cells were then washed and starved of methionine for 45 min with methionine-free RPMI (Thermo Fisher) supplemented with methionine-free FBS (Thermo Fisher). 2.5 μM AHA (Thermo Fisher) was then added back to the cells for 4 hr. Cells were harvested and counted by trypan blue exclusion, then cell pellets were frozen at −80°C until use. Total protein was labeled using the Click-IT protein labeling kit (Thermo Fisher) after normalizing the protein concentrations of cell lysates using a micro-BCA protein quantitation kit (Thermo Fisher). After labeling with 200 μM biotin-alkyne reaction buffer, protein was precipitated with chloroform/methanol extraction and resuspended in 35 μL of 2x sample buffer with β-mercaptoethanol, then heated for 7 min at 95°C. 5 μL of this sample was used for Silver stain (ProteoSilver by Sigma Aldrich), which was quantified by a Bio-Rad (Hercules, CA) GelDoc XR, and 30 μL used for Western blot, which was detected with streptavidin-A700 at 1:10,000 concentration by LICOR Odyssey.
4
Analyzing Viral Protein Synthesis in HeLa Cells
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The experiment was carried out using Click-iT AHA nascent protein kit (Thermo Fisher Scientific). Briefly, HeLa cells were cultured in a T-75 flask to a confluence of 95%. The cells were infected or mock infected with VACV at a MOI of 10. The cells were treated with DMSO or LY294002 (25 μM) at the desired times of infection. Cell culture medium was replaced with methionine-free medium at the indicated times. After incubation in methionine-free medium, AHA was added to the medium at 100 μM for 2 h. The cells were scraped off the flask and collected by centrifuging. Cell pellets were then suspended with 500 μl of lysis buffer containing 500U of benzonase for 30 min. After centrifuging at 12,000 g at 4°C for 10 min. The proteins were precipitated with methanol and chloroform and resolubilized in 50 mM Tris-HCl containing 1% SDS, pH 8.0. AHA-containing peptides were labeled with alkyne-biotin, by subjecting 200 μg of proteins to the click reaction for 30 min using the Click-iT protein labeling kit according to the manufacturer’s instruction (Thermo Fisher Scientific). Proteins were re-precipitated with methanol and chloroform, solubilized with 50mM Tris-HCl containing 1% SDS, pH 8.0, for Western blotting analysis.
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