Tgx stain free precast gel

Samples were separated by TGX stain-free precast gels (4-15% gradient, Bio-Rad), ultraviolet-activated with ChemiDoc Touch system (Bio-Rad) for direct imaging of total proteins and then transferred to nitrocellulose membranes for immunoblotting analysis. After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 136.7 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were incubated with rabbit anti-Nlg1 (129013, Synaptic systems) diluted at 1:1,000 with 0.5% non-fat dried milk in TBST, followed by horse radish peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch) for 1 h at room temperature. Target proteins were detected by chemiluminescence with Super signal West Dura (Pierce) on the ChemiDoc Touch system (Bio-Rad). The theoretical molecular weight of Nlg1 is 93 kDa, but the apparent molecular weight in immunoblots is ∼130 kDa, likely due to glycosylations65 (link). For quantification, the intensity of the chemiluminescence signal of each lane was normalized by the total protein signal on the same lane, revealed by the stain-free technology.

Protein samples were run on Bio-Rad TGX Stain-Free precast gels and visualized on the UV setting in the BioRad Chemidoc MP Imaging System to estimate total protein per lane. Blocking and antibody incubations were done in NAP blocker^TM (G-Biosciences). The mouse anti-VDR (Abcam) was used 1:10,000 and incubations were done overnight at 4°C. For detection, the blots were incubated overnight at 4 °C with anti-mouse biotin (Jackson Laboratories) followed by anti-mouse streptavidin-HRP (Vectastain Elite ABC system, Vector Laboratories). Molecular weight markers are Precision Plus Protein Kaleidoscope Standards (BioRad). Protein detection was achieved with femtoCHROMO^TM–HRP (G-Biosciences) and visualized using the colorimetric setting in the BioRad Chemidoc MP Imaging System.

To determine the oligomeric status of MtDef5 and its γ-core motif variants, protein cross-linking experiments were performed in the presence or absence of PIP, PA and PI cross-linked with bis[sulfosuccinimidyl] suberate (BS^{3 (link)}) substrate^{15 (link)}. Briefly, MtDef5 and its γ-core motif variants, prepared in 1X PBS buffer, at 1.5 mg/ml (5 µL) were incubated with 2.73 mM PI3P, PI4P or PI5P (1.5 µL) at room temperature for 30 min. The cross-linking reaction was initiated by addition of freshly dissolved water soluble 12.5 mM BS^{3 (link)} (0.5 μL) and incubated for 30 min at room temperature. After crosslinking, samples were reduced with 100 mM dithiothreitol (DTT) and separated on a 4–20% (Bio-Rad, TGX Stain-Free™ precast gels) SDS-PAGE. The oligomerization pattern was visualized using a Bio-Rad ChemiDoc XRS + system. For SDS-PAGE, Precision Plus Dual Xtra Protein Standards (2–250 kD) from Bio-Rad (Hercules, CA) were used.