Blue native page novex bis tris gel system

HLA-E-β2m complexes previously refolded with the UV-sensitive VL9 peptide (VMAPJTVLL) were incubated in the presence of molar excess test peptide and evaluated via the Blue Native-PAGE Novex Bis–Tris gel system (life technologies) (Walters et al., 2018 (link)). In brief, pre-refolded and purified HLA-E in complex with the UV-sensitive peptide was incubated for 3 h on ice in the presence of 12 M excess test peptide prior to the addition of 3 μL 4× Native-PAGE Sample Buffer per 10 μg (10 μL) of sample. Samples were loaded onto 4–16% Native-PAGE Novex Bis–Tris gels with NativeMark Unstained Protein Standard used as the ladder control. Gel electrophoresis was carried out at 150 Volts for 2 h at RT with a current gradient ranging from 15 to 16 to 2–4 mAmps. Gels were subsequently rinsed three times in MilliQ water and stained for 2–3 h in SimplyBlue SafeStain at RT. The MiliQ water was changed a number of times over a 24–48 h period to enable gel de-staining.

The composition of in vitro refolded HLA-E-β2m complexes was evaluated using the Blue Native-PAGE^™ Novex Bis–Tris gel system (life technologies), in accordance with the manufacturer’s instructions (https://tools.thermofisher.com/content/sfs/manuals/nativepage_man.pdf). In brief, 3 μL of 4× Native-PAGE^™ Sample Buffer was added to 10 μg (10 μL) of refolded HLA-E complexes, and immediately loaded on 3–12% Native-PAGE^™ Novex Bis–Tris gels. NativeMark^™ Unstained Protein Standard was used as the ladder control. Gel electrophoresis was performed at 150 Volts (with current gradient from 15–16 to 2–4 mAmps) for 2 h at room temperature. Following electrophoresis, gels were rinsed up to three times in MilliQ water prior to a 2–3 h staining step at room temperature in SimplyBlue™ SafeStain. De-staining was performed by multiple rounds of MilliQ water changes over a period of 24–48 h. Gel imaging was performed using a BioDoc IT Imaging System.