Horseradish peroxidase conjugated second antibody

Proteins in the resolved SDS-PAGE gel were transferred onto a PVDF membrane by electrophoretic transfer. The PVDF membrane was blocked with 3% skim milk in PBS for 1 h, and then the target proteins were probed using the specific antibody, anti-PTP1B (D-4, Santa Cruz biotechnology, Dallas, TX, USA), anti-IRS1 (E-12, Santa Cruz biotechnology, Dallas, TX, USA), or anti-IRS1 (phospho Y632, abcam, England and Wales, UK) antibody in 1% BSA overnight at 4 °C. After washing three times with PBS, for 10 min each time, the membrane was further incubated in horseradish peroxidase-conjugated second antibody (PerkinElmer, Waltham, MA, USA) for 1 h. The membrane was washed three times with PBS for 10 min each time, and the signals were detected using the standard ECL protocol (PerkinElmer, Waltham, MA, USA).

The PVDF membrane was blocked with 3% skim milk in PBS for 1 h and then the protein was recognized by the specific antibody, anti-GlxI (D-5, Santa Cruz biotechnology, Dallas, Texas, USA), anti-GlxII (A-11, Santa Cruz biotechnology, Dallas, Texas, USA), or anti-MG (Cell Biolabs) antibody, in 3 mg/mL of BSA overnight at 4 °C. After washed with PBS three times, each for 10 min, the membrane was further incubated in horseradish peroxidase-conjugated second antibody (PerkinElmer, Waltham, Massachusetts, USA) for 1 h. The signals were detected with the standard ECL protocol (PerkinElmer, Waltham, Massachusetts, USA) after washing the membrane with PBS three times, each for 10 min.

Proteins in the resolved SDS-PAGE gel were transferred onto a PVDF membrane by electrophoretic transfer. The PVDF membrane was blocked with 3% skim milk in PBS for 1 h, and then the target proteins were respectively probed by anti-CPTI (E-7), anti-CPTII (G-5), anti-HSP60 (H-1), and anti-Tom20 (29) antibody (Santa Cruz biotechnology, Dallas, TX, USA) in 1% BSA overnight at 4 °C. After washing with PBS thrice, each for 10 min, the membrane was further incubated in horseradish peroxidase-conjugated second antibody (PerkinElmer, Waltham, MA, USA) for 1 h. The membrane was washed with PBS three times, each for 10 min, and the signals were detected with the standard ECL protocol (PerkinElmer, Waltham, MA, USA).