Tris glycine sds page

RIPA buffer (Beyotime, Shanghai, China) with protease inhibitor was used to extract protein from tissue samples or cell lines. BCA quantitative assay (Beyotime) was used to measure the protein concentration. A total of 20 mg protein was separated by Tris-glycine SDS-PAGE (4–12%; Invitrogen) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). Blocking was performed by using 5% BSA for 2 h at room temperature. The membrane was incubated with primary antibodies overnight at 4°C. The following antibodies were used anti-CYBRD1 (HPA014757, Sigma-Aldrich), anti-IκBα (ab32518, Abcam, Cambridge, MA, USA), anti-p-STAT3 (ab267373, Abcam), anti-STAT3 (10253-2-AP, Proteintech, Wuhan, China), and anti-GAPDH (T0004, Affinity Biosciences, Cincinnati, OH, USA). Then, the membranes were incubated with the secondary antibodies (Cowin Biotech Co, Beijing, China) for 2 h at room temperature. Protein accumulation was detected by Western-light Chemiluminescent Detection System (Peiqing, Shanghai, China).

Total protein was extracted from hiPSC-RPE cells using RIPA buffer (ThermoFisher) and 1x protease inhibitor (Roche). Protein concentration of cell lysates was determined using the Direct Detect Assay-free cards (MerkMillipore, Australia), and 30 μg of protein from each lysate was resolved by 4-20% Tris-glycine SDS-PAGE (ThermoFisher, Australia). Protein was transferred onto a nitrocellulose membrane, which was blocked with 5% skim milk in 1x TBS and then incubated with rabbit anti-RPE65 (PA5-78414, ThermoFisher, USA) and mouse anti-β-actin (Sigma) antibodies, followed by fluorescent dye conjugated secondary antibodies (anti-rabbit IRDye 800CW and anti-mouse -IRDye 680RD, Millennium Science, Australia). Fluorescence detection was made using the ChemiDoc MP Imaging System (Bio-Rad, Australia), and acquired images were exported to Image Lab 6.0 software for quantification of RPE65 65 KDa protein band intensities normalised to β-actin levels (loading control).

Analytical gel filtration was performed on a Superose 6 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with Buffer A. The column was calibrated using carbonic anhydrase (29 kDa), alcohol dehydrogenase (150 kDa) and apo-ferritin (443 kDa) (Sigma-Aldrich) as molecular-weight standards. Unless otherwise indicated, 100 µl of each protein was injected at a concentration of 25 µM. Where indicated, fractions were collected and examined by 4–20% Tris–Glycine SDS-PAGE (ThermoFisher Scientific) and stained using InstantBlue coomassie Stain (Expedeon) or by Western blot analysis following transfer to a Protran BA85 nitrocellulose membrane (Whatman), blocking for 1 h with 0.5% skim milk, and visualized with anti-MMAB (1:1000, HPA039017 Sigma-Aldrich), anti-mouse HRP (1:5000, ab131368 Abcam) and ECL Western blotting Detection Reagent (GE Healthcare).