HEK293T cells were grown at 37°C, 5% CO2 in DMEM supplied with 10% FBS and 1% penicillin/streptomycin (all from Thermo Fisher Scientific, USA). Upon reaching 70% confluency, HA- and Strep-tagged constructs were transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the supplier’s recommendation. Cells were lysed after 48 hours with lysis buffer [150 mM KCl, 0.5% CHAPS, and 50 mM Hepes (pH 7.4)] containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, USA). Lysates were centrifuged at 3000g for 10 min at 4°C. Supernatants were collected and incubated with 1 U/ml of avidin to block endogenous biotin for 15 min at 4°C. Afterward, samples were centrifuged again at 3000g for 10 min at 4°C. Protein concentration was measured by analyzing absorbance at 280 nm and was set to 5 mg/ml. Strep-tagged protein was purified from 10 mg of total protein using 1-ml Strep-Tactin columns (IBA Lifesciences, Goettingen, Germany). The purification procedure was carried out as recommended by the supplier, and purified protein was eluted with 5 mM desthiobiotin. The eluate was analyzed by immunoblotting and silver-stained SDS-PAGE.

SDS gels were submitted to the Cambridge Centre for Proteomics, University of Cambridge. The protein bands were excised, containing proteins that were reduced, alkylated, and subsequently digested enzymatically. Samples were analyzed by reverse-phase high-performance liquid chromatography tandem MS. Resulting spectra were analyzed using the MASCOT database and interpreted using the UniProt database.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.