Anti β tubulin rabbit polyclonal antibody

Tissue samples were lysed in RIPA (Radio Immunoprecipitation Assay) Lysis Buffer (YEASEN, Shanghai, China) and centrifuged at 12,000× g at 4 °C. The protein concentration was determined using the BCA Protein Quantification Kit (YEASEN, Shanghai, China). Protein samples were separated in tris-glycine SDS-PAGE (12%) gel and transferred onto PVDF (polyvinylidene fluoride) membranes (300 mA for 20 min). The membrane was blocked overnight at 4 °C with 5% nonfat milk in TBST (Tris-Buffered Saline Tween-20), and then incubated for 2 h at room temperature with anti-PcIAG polyclonal antibody (prepared by Dia-An Biotech, Inc., Wuhan, China) (1:1000 dilution) targeting the full-length PcIAG ORF (Open Reading Frame) sequence, and anti-β-tubulin rabbit polyclonal antibody (Abcam, London, UK) (1:5000 dilution). After washing three times in PBST (phosphate buffered solution containing Tween-20), the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibodies (LI-COR, Lincoln, NE, USA) (1:10000 dilution) at room temperature for 2 h in the dark. After washing, the membrane was analyzed using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).

3 mg of Protein G Dynabeads (Life Technologies) were washed three times with 200 μl of PBST. After washing, 24 μg anti-β-tubulin rabbit polyclonal antibody (Abcam) was diluted in 0.1% PBST (200 μl) and incubated with the washed Dynabeads at 37°C for one hour with shaking at 1400rpm. Beads were washed three times with 200 μl of PBST, then conjugation buffer (20 mM Sodium Phosphate, 0.15 M NaCl, pH = 7–9) for crosslinking. Antibody was crosslinked to the Dynabeads using 250 μl of a freshly prepared solution of 5mM Bis (Sulfosuccinimidyl) substrate (BS³, Thermo Scientific) and incubated at room temperature for 30 min with shaking at 1400 rpm. The reaction was quenched by adding 1 M Tris HCl, pH = 7.5 (12.5 μl) for 15 min at room temperature. Beads were subsequently washed three times with 200 μl of 0.1% PBST.