Halotag alexa fluor 660 ligand

pMSCV-Halo-3× TEV-2× StrepII-PTB-puro and VSVg were co-transfected into the Ecotropic Phoenix HEK293T cells using Lipofectamine® 2000 Transfection Reagent (catalog No.11668019; Invitrogen) according to the manufacturer’s instruction. 48–72 h post transfection, media containing viruses were collected and filtered before infecting a HEK 293T cell line. 72 h after infection, puromycin (1 μg/ml) was added for the selection of stable cell lines. To check HaloTag fusion protein expression, approximately one million cells were collected and lyzed in 100 μl lysis buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM DTT, 1% Triton X-100, 10% glycerol, and 1× protease inhibitor cocktail (catalog No. G6521; Promega) on ice for 15 min. The insoluble fractions were removed by centrifugation at maximum speed at 4 °C for 10 min using Centrifuge 5424R (Eppendorf) and the supernatant was incubated with HaloTag® Alexa Fluor® 660 Ligand at 1 μM final concentration (catalog No. G8471; Promega) at room temperature for 15 min. Then samples were heated with 4× SDS gel loading buffer at 70 °C for 10 min and loaded onto SDS–PAGE gels. After electrophoresis, the SDS–PAGE gels were directly scanned using the Odyssey® CLx Imaging System at 700 nm.