Odyssey infrared imaging blocking buffer

RBL cell proteins were extracted by lysis with 1% Triton X-100, 1% NP40, 10% glycerol, 137 mM NaCl, 20 mM tris HCl, 1 ng/μl aprotinin, 1 ng/μl leupeptin, 4 mM PMSF, 20 mM NaF, and 1 mM Na₃VO₄. Samples were prepared by addition of 4× lithium dodecyl sulfate buffer supplemented with DTT to a final concentration of 50 mM, heating for 10 min at 70 °C, followed by electrophoresis on 4–12% bis–tris gels (Invitrogen, Carlsbad, CA) in 1× MES-SDS buffer. Proteins were electrophoretically transferred to PVDF membranes and blocked in Odyssey infrared imaging blocking buffer (Licor, Lincoln, NE). Mouse anti-phospho-Akt Ser473, rabbit anti-total Akt, and rabbit anti-phospho-STAT3 Ser727 antibodies were obtained from Cell Signaling (Danvers, MA). Mouse anti-phospho-Pyk2 Tyr 402 antibody was from Santa Cruz (Santa Cruz, CA). Primary antibodies were diluted in the Odyssey blocking buffer supplemented with 0.1% Tween 20 and incubated overnight at 4 °C. Proteins were visualized by incubation of membranes with either fluorescent-tagged anti-mouse and anti-rabbit antibodies (Molecular Probes, Carlsbad, CA) and scanning on an Odyssey Infrared Imager (Licor), or, incubation with HRP-linked secondary antibodies and use of enhanced chemiluminescent substrate (Pierce, Rockford, IL).