Nativepage 4 16 bis tris gel

A modification of the BN-PAGE method was performed in the presence of the indicated concentration of the reducing agent DTT and/or with sample heating at the indicated temperature prior to electrophoresis. Native PAGE 4–16% BisTris gels (Invitrogen) were used following the manufacturer's instructions. The indicated E2 antigens (4 μg) and NativeMark protein standards (Thermo Fisher Scientific) were adjusted to 1× sample buffer (4× sample buffer: 200 mm BisTris, 64.2 mm HCl, 200 mm NaCl, 40% (w/v) glycerol, 0.004% (w/v) Ponceau S) prior to loading on the gel. The gel was fixed in 50% ethanol and 2% phosphoric acid; stained in 8.5% phosphoric acid, 10% ammonium sulfate, 20% methanol, and 0.12% Coomassie blue G-250 dye; and imaged using a LI-COR Odyssey IR imager and version 3.0 software. Band intensity was quantified with Image Lab version 6 software (Bio-Rad). Denaturing SDS-PAGE of the indicated E2 monomeric antigens (4 μg) and Precision Plus protein standards (Bio-Rad) was performed using standard conditions either in the absence or presence of the reducing agent, β-mercaptoethanol. Gels (12% Tris/glycine) were stained with Coomassie dye and imaged as above.

Thylakoids were prepared as described by Jarvi et al. (2011 (link)), using 5 × 5 mm leaf discs of infiltrated leaf areas per sample. Pelleted thylakoids were suspended in 40 μl native PAGE buffer (Invitrogen) with 1 % n-dodecyl-β-d-maltoside (Invitrogen) on ice for 15 min, centrifuged for 15 min at 4 °C, and the supernatants removed and stored at −80 °C. Prior to loading gels, 0.75 μl 5 % G-250 sample additive was added to 15 μl aliquots of thylakoid protein extracts. Electrophoresis was carried out using NativePAGE™ 4–16 % Bis–Tris gels (Invitrogen) at 150 V. For two-dimensional analysis, lanes from native PAGE were excised and proteins denatured in LDS sample buffer containing 0.05 M DTT for 30 min. Proteins in gel slices were analysed by electrophoresis using 12 % SDS Bis–Tris two-dimensional well gels (Novex, Life Technologies). Identification of bands was based on comparisons with earlier NativePAGE analytical data (Liu and Last 2015 (link)).

Non-denaturing Blue-Native PAGE was performed to investigate the native structure of ApoE complexes. Astrocyte and neuron media samples, and recombinant ApoE samples were mixed with NativePAGE^TM Sample Buffer (Invitrogen, BN2003). The samples were loaded onto a NativePAGE^TM 4–16% Bis-Tris gel (Invitrogen, BN1002BOX) and run at 150 V. NativePAGE running buffer (Invitrogen, BN2001) and NativePAGE^TM Cathode Buffer Additive (Invitrogen, BN2002) were used as running buffers. NativeMark^TM Unstained Protein Standard (ThermoFisher Scientific, LC0725) was run on the gel as molecular weight marker and was visualized, after protein transfer, using Ponceau C. After protein transfer, the membranes were boiled two times in PBS for 5 min, followed by 7 min incubation in 10% formic acid, prior to blocking, according to a protocol previously described by Heinsinger et al. (2016) (link).

NativePAGE^TM 4–16% Bis-Tris gel (Invitrogen) was used for clear native electrophoresis. Protein samples in the presence of 0.02% β-DDM were mixed with 2X native sample buffer (100 mM sodium chloride, 100 mM imidazole-HCl, 4 mM 6-aminohexanoic acid, 10% glycerol and 2 mM EDTA, pH 7.0) [41 (link)]. The cathode buffer contains 50 mM Tricine, 7.5 mM imidazole, pH 7.0 and the anode buffer contains 25 mM imidazole, pH 7.0 [42 (link)].
The relative mobilities (R_f) of protein standards were plotted against the known molecular mass (MW) values of the standards. Nonlinear regression was used to obtain the following formula: MW = 2054e^-3.6Rf-65 (R² = 0.99); the formula in turn was used to interpolate the apparent molecular masses associated with the proteins bands.
The relative mobilities (R_f) of protein standards were plotted against the known molecular mass (MW) values of the standards. Nonlinear regression was used to obtain the following formula: MW = 2054e^-3.6Rf-65 (R² = 0.99); the formula in turn was used to interpolate the apparent molecular masses associated with the proteins bands.