The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). A 52 kDa band corresponding to alpha Tubulin was observed across the cell lines and tissue tested. The blot was probed with Anti-alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-29546, 1:2000) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), C2C12 (Lane 4), MDA-MB-231 (Lane 5), PC-12 (Lane 6), RSC96 (Lane 7) and tissue extract of Mouse Lung (Lane 8).
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