Alpha Tubulin Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Rabbit (1:5000) -Soak PVDF membranewith pore size 0.45 um in methanol for 30 sec -Apply Wet transfersystems at 180mA for 90 min with Wet transfer buffer.

Protocol tips
Rabbit (1:5000) -Soak PVDF membranewith pore size 0.45 um in methanol for 30 sec -Apply Wet transfersystems at 180mA for 90 min with Wet transfer buffer.

Publication protocol

Cells were harvested using lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (PH7.4), 1% Nonidet P-40, 0.5 mM EDTA, PMSF (50 μg/ml) and protease inhibitors mixtures (Sigma). N-ethylmaleimide (10 mM) was added into the above lysis buffer when protein ubiquitination was detected. Equivalent protein quantities were subjected to SDS-PAGE or native-PAGE, and transferred to PVDF membranes. Membranes were then blocked with 5% non-fat milk or 5% BSA for 1 hr at room temperature and then probed with the primary antibodies, followed by the respective HRP-conjugated Goat anti-mouse or Goat anti-rabbit (Bioworld) secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended kits (Thermo Scientific). The following antibodies were used: antibodies against pY701-STAT1 (1:1,000, Cell Signaling, #9167), pS727-STAT1 (1:1000, cell signaling, #8826), pY690-STAT2 (1:1000, Santa Cruz, sc-21689-R), STAT1 (1:2,000, Cell Signaling, #9172), Ubiquitin (1:1,000, Santa Cruz, sc-8017), VSV-G (1:5,000, Santa Cruz, sc-66180), HA (1:2,000, abcam, ab9110), K48Ub (1:500, Cell Signaling, #4289), K63Ub (1:500, Millipore, #05–1308), USP2 (1:1,000, ABGENT, AP2131c), Flag (1:5,000, Sigma, F7425), Smurf1 (1:1000, Santa Cruz, sc-100616), β-Actin (1:5,000, Proteintech, #66009), KPNA1/Importin α5 (1:1,000, Santa Cruz, sc-101292), Lamin B1 (1:2,000, Proteintech, 12987-1-AP), Alpha-Tubulin (1:5,000, Proteintech, 66031-1-Ig). The Image J program (http://rsbweb.nih.gov/ij/download.html) was used for densitometric analysis of western blots.

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Manufacturer protocol

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