Amersham hybond p polyvinylidene difluoride pvdf membranes

Gels were transferred to Amersham Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare) at 400 mA for 2 h. In order to confirm the correct transference of the proteins, membranes were stained with Ponceau Red (0.2% (p/v) Ponceau Red, 1% (v/v) acetic acid). For antigen detection, membranes were blocked in Tris-Buffered Saline (TBS) plus 5% (p/v) skimmed milk and 0.1% (v/v) Tween 20 (TBSM) for 2 h. Then they were incubated overnight with mouse serum diluted in TBSM at 4 °C. After that, membranes were washed four times for 5 min with TBS and then incubated with murine anti-IgG-HPR diluted to 1/100,000 in TBSM. All the incubations were made at room temperature, unless otherwise stated. Immunoreactive proteins were detected using ECL Plus (GE Healthcare) following the manufacturer instructions in the G:BOX Chemi system (Syngene). Western Blot (WB) analysis was carried out using ImageMaster 2D Platinum Software (GE Healthcare).

Total cell lysates were extracted by Upstate Mg²⁺ lysis buffer (Merck Millipore, Darmstadt, Germany), and then dissolved in 7~12% gels of sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis (SDS-PAGE). The proteins were then transferred onto Amersham Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare Life Science, Piscataway, NJ, USA). Indicated proteins were hybridized with primary antibodies in 3% BSA solution, washed with tris-buffered saline containing 0.1% tween 20, and then probed with horseradish-peroxidase (HRP)-labeled secondary antibodies. The emitted signal of target proteins was developed by chemiluminescence reagent plus (PerkinElmer Life Sciences, Boston, MA, USA) and identified by BioSpectrum Imaging System (UVP, LLC Upland, CA, USA).

Total cell lysates were extracted by RIPA lysis buffer (Merck Millipore, Darmstadt, Germany), and then dissolved in 7–12% gels of sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis (SDS-PAGE). The proteins were then transferred onto Amersham Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare Life Science, Piscataway, NJ, USA). The indicated proteins were hybridized with primary antibodies in 3% BSA solution, washed with tris-buffered saline containing 0.1% tween 20, and then probed with horseradish-peroxidase (HRP)-labeled secondary antibodies. The emitted signal of target proteins was developed by chemiluminescence reagent plus and identified by a BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA).