This assay was performed as described18 (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).
Click it protein reaction buffer
Manufactured by Thermo Fisher Scientific
Click-iT® Protein Reaction Buffer is a reagent used in the Click-iT® protein labeling technology. It is designed to facilitate the conjugation of azide- or alkyne-modified proteins with their respective clickable partners. The buffer provides the necessary components to enable the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, which is a common method for protein labeling and detection.
Automatically generated - may contain errors
Lab products found in correlation
Methionine free dmem, by Thermo Fisher Scientific (2 mentions)
Click it hpg, by Thermo Fisher Scientific (2 mentions)
Protein g agarose, by Merck Group (2 mentions)
Click it protein labeling kit, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
L azidohomoalanine aha, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Dynabead, 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)
Dynabeads m 280, by Thermo Fisher Scientific (1 mentions)
4 protocols using click it protein reaction buffer
1
Analysis of de novo protein synthesis
2
Metabolic Labeling of Nascent Proteins
3
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.
4
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.
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