Phosbind reagent

HEK293T cells from a near confluent 10 cm dish were harvested, pelleted by centrifugation, and resuspended in 500 μl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630) supplemented with protease inhibitor cocktail (Goldbio, GB-331). Following incubation on ice for 15 minutes, extracts were cleared by centrifugation. Phosphatase reactions (100 μl total volume) containing cleared lysates supplemented with MnCl₂ (1 mM final concentration), 100 units lambda phosphatase (NEB, P0753S), and/or phosphatase inhibitor cocktail (2x final concentration) (Sigma, 4906837001) were prepared on ice and subsequently incubated at 30 °C for 1 hour. Reactions were then boiled with LDS sample buffer (Thermo Fisher, NP0008) supplemented with beta-mercaptoethanol, resolved by SDS-PAGE using a 6% acrylamide gel supplemented with 75 μM PhosBind reagent (APExBIO, F4002) and 150 μM MnCl₂, and transferred to nitrocellulose membrane for immunoblotting.

HEK293T cells from a near-confluent 10 cm dish were harvested, pelleted by centrifugation and resuspended in 500 µl lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630) supplemented with protease inhibitor cocktail (Goldbio, GB-331). Following incubation on ice for 15 minutes, extracts were cleared by centrifugation. Phosphatase reactions (100 µl total volume) containing cleared lysates supplemented with MnCl₂ (1 mM final concentration), 100 units lambda phosphatase (NEB, P0753S), and/or phosphatase inhibitor cocktail (2x final concentration) (Sigma, 4906837001) were prepared on ice and subsequently incubated at 30 °C for 1 hour. Reactions were then boiled with LDS sample buffer (Thermo Fisher, NP0008) supplemented with β-mercaptoethanol, resolved by SDS-PAGE using a 6% acrylamide gel supplemented with 75 µM PhosBind reagent (APExBIO, F4002) and 150 µM MnCl₂, and transferred to nitrocellulose membrane for immunoblotting.