Bis tris mini protein gel

Characterization of human hair keratin was performed by SDS-PAGE analysis.100 μg of human hair keratin was dissolved in 13 μl of DBPS and mixed with 5 μl of LDS (Invitrogen, B0007). and 2 μl of sample reducing agent (Invitrogen, B0009). Each sample was denatured by heating at 75 °C for 10 min before loading into precast Bolt 4 to 12% Bis-Tris Mini Protein Gels (Invitrogen, NW04122BOX). Electrophoresis was carried out at a constant voltage of 200 V for 22 min in 1× MES Running Buffer (Invitrogen, B0002). Subsequently, separated proteins were stained with SimplyBlue SafeStain (Invitrogen, LC6060).

S. cerevisiae strains expressing constructs tagged with 3×FLAG-6×His were grown overnight in liquid synthetic complete medium with 2% galactose (Formedium, Norfolk, UK), diluted to an OD of 0.1, and cultured again to exponential phase (OD of 0.5). For protein extraction, approximately 10⁸ cells were washed with sterile water and suspended in 200 μL lysis buffer containing 0.1 M NaOH, 0.05 M EDTA, 2% SDS, and 2% β-mercaptoethanol. Cells were then boiled at 90°C for 10 min, 5 μL of 4 M acetic acid was added, and the sample was boiled again for 10 min. Concentration was measured with the Qubit protein assay following the manufacturer instructions, and ~50 μg of the protein was loaded to 8% bis-tris mini protein gels (Invitrogen Bolt) with a 0.25 M Tris-HCl (pH 6.8), 50% glycerol, and 0.05% bromophenol blue loading buffer. Electrophoresis using bis-tris gels was performed according to manufacturer’s instructions with morpholinepropanesulfonic acid (MOPS) buffer, and proteins were transferred to a nitrocellulose membrane. The 6×-His tag monoclonal antibody (HIS.H8; Invitrogen) and goat anti-mouse IgG (H+L) horseradish peroxidase (HRP) (Invitrogen) were used to probe the membrane as primary and secondary antibodies, respectively. Detection was done using the Novex ECL chemiluminescent substrate reagent kit (Invitrogen).

NSCs were lysed by RIPA buffer supplemented with a protease inhibitor cocktail (Thermo Scientific). The total protein samples were loaded to run SDS-PAGE on the NuPAGE 4 to 12%, Bis-Tris mini protein gel (Thermo Fisher, cat# NP0335BOX), and transferred to the nitrocellulose membrane (Thermo Fisher, cat# LC2006). After blocking in 5% milk-PBST, the membrane was incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 1h, with washing steps in between. The membrane was developed with Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, cat# 34580), and the images were captured using the Bio Rad ChemiDoc imaging system. The primary antibodies used were rabbit anti-Ndufa13 antibody (Proteintech, cat# 10986-1-AP), rabbit anti-Gng11 antibody (Thermo Fisher, cat# PA5-100666), mouse anti-Ptx3 antibody (Santa Cruz, cat# sc-373951), and rabbit anti-GAPDH antibody (CST, cat# 2118).

Total protein was extracted from 293T and HepG2 cell lines by RIPA buffer (Thermo Fisher) containing 1× protease inhibitors (Thermo Fisher). 20 μg total proteins of each sample were loaded into a 4 to 12% Bis-Tris mini protein Gel (Thermo Fisher). Subsequently, proteins were transferred to a 0.2 μm PVDF membrane (BioRad). After blocking in 5% non-fat milk at room temperature for one hour, membranes were incubated with rabbit anti-human ENPP1 antibody at 1:1000 dilution (Abcam, EPR22262-75) or β-Actin antibody at 1:1000 (Cell signaling technology) for 4°C overnight. After three washes with TBST, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (Thermo Fisher) at 1:3000 dilution for 1h at room temperature. After 5 additional washes with TBST, the membranes were visualized by using ECL western blot substrate (Thermo Fisher) and imaged by ChemiDoc XRS+ imaging system (Bio-Rad).