Supersignal molecular weight protein ladder

Protein samples were loaded into a 10% Bis–Tris NuPAGE gel and electrophoresed in SDS-MES buffer alongside a protein molecular weight standard; EZ-Run™ Prestained Rec Protein Ladder (Fisher BioReagents) or SuperSignal molecular weight protein ladder (Thermo). For protein concentration determination, samples of unknown concentration were loaded at multiple dilutions and electrophoresed alongside a V565 reference standard curve. The gels were imaged in an Imagequant LAS 4000 (Cytiva) using the white transmitted light table and analysed using Imagequant TL software to determine unknown protein concentrations from the V565 standard curve.

The total protein of cultured control and treated cells were extracted based on SDS, and the concentration of isolated protein was determined using the Bradford method (He, 2011[26 ]). Subsequently, denatured proteins, as the result of boiling in 100 °C water, were loaded on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels with a protein ladder (SuperSignal® Molecular Weight Protein Ladder, Thermo Fisher Scientific, USA). Separated proteins were transferred to the PVDF (polyvinylidene fluoride) membranes to isolate the target protein in a western blotting device; then the membranes were incubated with primary antibodies β-actin (sc-47778,1: 300), GAPDH (sc-47724, 1:300), and Glu synthetase (E-4) (sc-74430, Santa Cruz Biotechnology, 1:300) for 16-18 hours at 4 °C. Followingly, the PVDF membranes were washed with TBS-T buffer and shaken in a solution; containing secondary antibodies (anti-rabbit, 1:1000) for 75 minutes. Finally, the labeled proteins were visualized and investigated by chemiluminescence assay (ECL advanced reagents kit, Amersham, USA). The collected raw data were analyzed using Student's t-test. P value < 0.05 was considered statistically significant.

To confirm the presence of CobC in liposome flotation mixtures and to analyze proteolytic digests, dot blots were performed using rabbit polyclonal antibodies generated against CobC (Envigo, Indianapolis, IN). For dot blot analysis, 3 μL of 1 mM proteoliposomes obtained by liposome flotation and 100 ng of positive controls was spotted onto a nitrocellulose membrane. Membranes were incubated for 30 min in blocking buffer of phosphate-buffered saline containing Tween 20 (PBST) comprised of NaH₂PO₄ (10 mM, pH 7.2), NaCl (0.9% [wt/vol]), Tween 20 (0.1% [vol/vol]), and instant dry milk (5% [wt/vol]). Membranes were probed with anti-CobC or anti-CobS antibodies (1:5,000 in blocking buffer) for 1 h, then washed three times (30 min each) with PBST. Membranes were probed for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Sigma) in PBST (1:10,000) before three 30-min washes with PBST. Membranes were incubated in SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher) for 5 min and imaged using a UVP ChemStudio imaging instrument (Analytik Jena). Purified CobC protein was used as a positive control, and a SuperSignal molecular weight protein ladder (Thermo Fisher) was used as a reference for the electrophoretic behavior of molecules of known molecular masses.