Anti gst antibody

The brain samples (penumbral cortex) were collected at 24 h post-stroke and were used for Western blot analysis according to previously described method [22 (link)]. Primary antibodies used in the studies include anti-Ubqln1 (mouse) antibody (1:1000, Abcam), anti-Ubqln1 (human) antibody (1:1000, Life Technology), anti-GST antibody (1:1000, Cell Signaling), anti-polyUb antibody (1:1000, Cell Signaling), anti-K48-linked polyUb antibody (1:1000, Cell Signaling), and anti-actin (1:1000, Santa Cruz Biotechnology). Detection was performed using appropriate secondary antibodies conjugated with the infrared dyes (1:5000, LI-COR Inc., Lincoln, NE, USA). Protein band intensities were measured using an Odyssey scanner (LI-COR) and quantified using UN-SCAN-IT gel 6.1 software (Silk Scientific Inc., Orem, Utah, USA).

After printing, SiNW substrates were blocked using SuperBlock TB (Thermo Scientific, Waltham, MA) for 30 min. The substrates were rinsed, dried and placed in an airtight chamber [18 (link)] with a flexible film above the substrates. Air in the chamber was removed by vacuum from a fluid gap between the substrate and the flexible film. Human HeLa cell lysate-based IVTT system was injected into the fluid gap by syringe thus filling nanowells with lysate. Excess reagent was swept away from the substrate by flowing pressurized viscous liquid over the flexible film. Individual nanowells were thus sealed into isolated reaction chambers by the flexible film and pressurized viscous liquid. The chamber was placed in an incubator (EchoTherm chilling incubator, Torrey Pines Scientific, Carlsbad, CA) for protein expression at 30°C for 2 hrs and capture at 15°C for 1 hr. Displayed proteins were detected with a monoclonal anti-GST antibody (Cell signaling Inc., Danvers, MA) and Alexa Fluor® 647 goat anti-mouse IgG (H+L) (Life technologies, Carlsbad, CA). Substrates were washed, dried and scanned by Tecan PowerScanner^™ (Tecan Group Ltd, Männedorf, Switzerland).