Click go protein reaction buffer kit

Azidohomoalanine-based pulse-chase assay was performed according to a previous protocol (Wang et al., 2017 (link)). Cells were plated and transfected in 6-cm dishes. Pulse-chase was performed by incubating the cells in methionine/cysteine/glutamine-free DMEM with 10% dialyzed FBS, 1× GlutaMax, 0.2 mM L-cysteine, and 50 μM L-azidohomoalanine for 1 hr at 37°C. The medium was then replaced by the normal cell culture medium supplemented with 2 mM L-methionine for 0–3 hr at 37°C for the chase. The cells were then lysed in the sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 100 mM Tris-HCl pH 8.0 and 1× protease inhibitor cocktail in water) for 30 min at 4°C, and then centrifuged at 16,000 g for 15 min at 4°C.
Labeling of the incorporated AHA by biotin-alkyne was conducted using the Click-&-Go Protein Reaction Buffer Kit (Click Chemistry Tools) according to the manufacturer’s protocol. The reacted mixture was dialyzed in PBST buffer (DPBS with 0.05% v/v Tween 20) by a centrifugal filter with a 3 KDa molecular weight cut-off. The supernatant was immunoprecipitated by custom-made anti-RFP beads (see Immunoprecipitation [IP] and Co-IP below). Biotinylated proteins were detected by western blotting using NeutrAvidin-Alexa Fluor 647.

One day after seeding 300, 000 cells/well, cells were infected with AdSLFN12 or AdCMV. Sixty hours later, the medium was replaced with methionine-free medium (Thermo Fisher, #21013024) for two hours. Cells were incubated with 50μM L-Azidohomoalanine (AHA) (Clickchemistrytools, Scottsdale, AZ, #106625) for 8 hours and lysed in NP-40 lysis buffer (Thermo Fisher, #FNN0021) with 1%SDS. AHA was detected using click chemistry with 50μM Biotin Alkyne (Clickchemistrytools, #1266–5) with Click-&-Go^™ Protein Reaction Buffer Kit (Clickchemistrytools, #1262) per manufacturer’s protocol. After click chemistry, ZEB1 was immunoprecipitated using rabbit anti-ZEB1 and protein-A magnetic beads (Bio-Rad Laboratories, #161–4013). Proteins were resolved by 10% SDS-PAGE, transferred to 0.45μm nitrocellulose membrane, and blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, #927–50010). Biotin was labeled with IRDye 800CW Streptavidin per manufacturer’s protocol (LI-COR, #926–32230) and ZEB1 was labeled with IRDye 680LT Donkey anti-Rabbit IgG Secondary Antibody. Images were acquired using a LI-COR-Clx and analyzed using Image Studio (LI-COR).