Goat anti gfp antibody

For the elucidation of phosphorylation status, phos-tag SDS-PAGE, which is capable of separating phosphorylated and non-phosphorylated proteins based on phosphorylation levels by capturing phosphate residues, was used. Phos-tag gels were purchased from Wako. Cell lysates or fractionated samples were used for western blotting. To obtain dephosphorylated protein as a completely dephosphorylated control, lambda protein phosphatase (New England Biolabs) treatment was performed prior to SDS-PAGE. Each fraction or cell lysate was mixed with Laemmli sample buffer (Bio-Rad), resolved on SDS-PAGE or phos-tag SDS-PAGE, and transferred to PVDF membranes. Protein-bound membranes were blocked with 5% skim milk buffer and incubated with primary antibody diluted in 0.5% skim milk buffer. Mouse anti-beta-actin antibody (1:5000, Sigma-Aldrich), rabbit anti-Y14 antibody (1:2000, Sigma-Aldrich), goat anti-GFP antibody (1:2000, Santa Cruz Biotechnology), and mouse anti-fibrillarin antibody (1:1000) were used as the primary antibody. Binding of primary antibody was detected with horseradish peroxidase-conjugated anti-mouse IgG, anti-goat IgG, and anti-rabbit IgG antibodies (Agilent Technologies). The membrane was subsequently washed and developed with ImmunoStar Zeta chemiluminescent reagents (Wako) and chemiluminescence was visualized using the LAS4000 (Fujifilm).

NSC-34 cells were lysed in Radio Immunoprecipitation Assay (RIPA) (ThermoFisher) buffer with protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Scientific). Then, 30 µg of proteins were separated by SDS-PAGE in a 4–15% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was treated with 5% of milk in Tris-buffer containing 0.1% Tween-20 before overnight incubation at 4 °C with a polyclonal goat anti-GFP antibody (sc-5385, 1/200 Santa Cruz Biotechnology^® Inc., Dallas, TX, USA). Horseradish peroxydase-conjugated rabbit anti-goat antibody (81-1620, 1/2500, Zymed, South San Francisco, CA, USA) was used as secondary antibody before chemiluminescence analysis using ECL (Pierce-Thermo Fischer Scientific Inc., Rockford, IL, USA) and quantification by QuantityOne software (BioRad, Hercules, CA, USA). GAPDH expression was used for normalization using polyclonal goat anti-GAPDH antibody (sc-48166, 1/250, Santa Cruz Biotechnology Inc.) and secondary donkey anti-goat antibody (V8051, 1/2500, Promega, Madison, WI, USA).