Nupage novex bis tris gel 1.0 mm

Protein samples were separated by SDS-PAGE electrophoresis (4–12% gradient, NuPAGE Novex Bis-Tris Gel 1.0 mm, Life Technologies) and transferred onto Protran Premium membrane (nitrocellulose, GE Healthcare). Fifty micrograms (1 µl of serum) of human, mouse or dog serum protein were loaded per lane. Antibodies against MYOM3 (1:1000, Proteintech: 17692-1-AP) and the CK-M (1:500, Santa Cruz: sc-15161) were used as primary antibodies followed by incubation with the corresponding IRDye-800CW-conjugated antibodies (1:10 000, LI-COR Biosciences) according to the manufacturer's instructions. Infrared fluorescence of the secondary antibodies was read on an Odyssey Imaging System (LI-COR Biosciences). Band intensities were measured by the Odyssey application software (LI-COR Biosciences, Image Studio Lite 4.0 Version). The following antibodies were not efficient in Western blot analysis of human serum: anti-Myomesin 3 sc-165061; anti-Myomesin 2: sc-30384; sc-30385; sc-50435; anti-Myosin: ABIN502241; ABIN616957; sc-53089; sc-20641; sc-32732; sc-53096.

Cell lysates were prepared from 4 hpf and 24 hpf UIC, wnt10a Tb‐ and SbMO‐injected embryos, and wnt10a RNA‐injected embryos, and dechorionated with pronase. After de‐yolking manually on an agarose plate, the embryos were collected and mixed with SDS sample buffer and boiled for 10 min. Proteins were resolved by SDS‐PAGE electrophoresis (4–12% gradient, NuPAGE Novex Bis‐Tris Gel 1.0 mm) (Life Technologies, Foster City, CA) and then transferred onto PVDF membrane (Invitrogen iBlot Dry, Carlsbad, CA). The membrane was incubated with primary antibodies anti‐wnt10a (cat. ARP41277_P050, LI‐COR Biosciences, Lincoln, NE) and antiactin (cat. Ab8227, Abcam, Cambridge, UK) at room temperature for 2 h. Following washing three times with 1xTBST for 15 min each, the membrane was incubated with IRDye^® 800 CW antirabbit IgG secondary antibody (cat. 926‐32211, LI‐COR) for 1 h at room temperature. Protein bands were visualized using LI‐COR Odyssey Scanning Imager (LI‐COR).