Click it protein reaction buffer kit

Newly translated proteins were labeled by floating leaf discs (4 mm) from the most mature (largest) leaves of 6-week-old wild type and amiNAA10 plants for 4 h on ½ Hoagland medium supplemented with 50 µM L-homopropargylglycine (HPG, Invitrogen, for biotin labeling of nascent proteins) or 50 µM L-azidohomoalanine (AHA, Invitrogen, for nascent protein enrichment). After the incubation medium was removed from the leaf discs and subsequently frozen in liquid nitrogen and stored at −80 °C.
To visualize the newly translated proteins, HPG labeled proteins were clicked to Biotin Azide (Invitrogen) using the Click-iT™ Protein Reaction Buffer Kit (Invitrogen) following the manufactures protocol. The biotin-labeled proteins were immunologically detected after separation of the protein crude extract by SDS-PAGE and blotting onto a PVDF membrane using Neutravidin-HRP (1:100,000, Invitrogen, 31001).
Nascent proteins were enriched by cycloaddition of AHA-labeled proteins to the alkyne agarose resin using the Click-iT™ Protein Enrichment Kit (Invitrogen) following the manufacturers protocol. Proteins were eluted by trypsin digestion from the resin after stringent washing with 1% SDS (5 times), 8 M Urea/100 mM Tris pH 8 (10 times) and 20% acetonitrile (10 times) to eliminate non-specifically bound proteins from the resin.

De novo protein synthesis assays were performed using the methionine analogue L-azidohomoalanine (AHA)^{60 (link)}. Briefly, cells were starved for 30 min in methionine-free RPMI media containing 10% dialysed FBS and 0.2 mM L-cysteine. Cells were given a pulse with AHA (100 µM) and incubated for 2 h at 37 °C in a humidified 5% CO₂ incubator. Cells were washed in PBS, harvested in lysis buffer (1% SDS in 50 mM Tris-HCl pH 8.0) and boiled at 95 °C or 50 °C for OXPHOS proteins. Biotin labelling was performed using the Click-iT Protein Reaction Buffer kit (Invitrogen, C10276) as per manufacturer’s protocol, followed by streptavidin pull-down (Dynabeads M-280 streptavidin) and western blot analysis.

Cells were uninfected or infected with SARS-CoV-2 for indicated times. Starvation of cells was done for 3 h by incubating with RPMI Medium 1640 without l-methionine (Thermo Fisher). Cells were then incubated with the same medium supplemented with the Click-IT-AHA (L-Azidohomoalanine) reagent (Invitrogen) for 2 h. Cells were lysed in a solution containing 1% SDS in 50 mM Tris-HCl, pH 8.0. Click-chemistry reaction for protein detection was performed using biotin alkyne (PEG4 carboxamide-propargyl biotin) according to manufacturer’s instructions using the Click-iT Protein Reaction Buffer kit (Invitrogen). Labeled proteins were separated by SDS-PAGE. The membrane containing the biotin alkyne labeled proteins was incubated in a solution containing 1% BSA with streptavadin-HRP (Invitrogen) for subsequent visualization.

The Click-iT® Protein Reaction Buffer Kit (Invitrogen, Grand Island, NY, USA) was used to assess the level of O-fucosylation. In brief, 6-alkynyl fucose (6AF) (50 μM) was added in the cell culture medium for 48 h. Lysate solution 200 μl (1% SDS, 50 mM Tris–HCl, pH 8.0) was added to the cell culture and lysed on ice for 30 min to collect cells. Click reactions were performed as on both the labeled cell lysate to tag glycoproteins containing 6AF with biotinylated azide at room temperature for 1 h. The tagged cell lysate was analyzed by immunoblot.

Newly synthesized protein was labeled with Click-IT Metabolic Labeling Reagents for Proteins (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions as described previously (59 (link)). Primary murine macrophages or HEK293T cells cotransfected with the indicated constructs were seeded and cultured in methionine-free medium for an hour. Cells were then cultured in methionine-free medium containing 50 μM of AHA (a methionine analog; Invitrogen) for 4 hours. After washing with PBS, the medium was changed with complete medium, and the cells were chased for the indicated time. The cells were lysed in the lysis buffer (1% SDS, 50 mM Tris-HCl [pH 8.0], and protease inhibitor), and AHA-incorporated protein was biotinylated using the Click-IT Protein Reaction Buffer Kit (Invitrogen, Thermo Fisher Scientific) and Biotin Alkyne (Invitrogen, Thermo Fisher Scientific). After precipitation and dissolving, biotinylated protein was collected with Dynabeads M-280 Streptavidin (Invitrogen, Thermo Fisher Scientific). The purified proteins were probed with the indicated antibodies for Western blot analysis.