Nupage 4 12 bis tris pre cast gels

Samples mixed with 2 × Laemmli sample buffer and 1 M dithiothreitol (95 °C, 10 min), and electrophoresed in NuPAGE 4–12% Bis-Tris Pre-cast gels (BioRad; Hercules, CA, USA) for 50 min at 150 V with Precision Plus Protein^TM Dual Color Standard (BioRad; Hercules, CA, USA) were dry-blotted using nitrocellulose membranes iBlot^® Gel Transfer Stacks (Invitrogen, Life Technologies; Carlsbad, CA, USA). Membranes were blocked by rolling in 5% skim milk in PBS-0.05% Tween-20 (Amresco; Solon, OH, USA) (RT, 1 h), followed by incubation in primary antibody from 1) pooled or individual (using a Mini-Protean Multiscreen apparatus (Biorad)) TB and non-TB sera at 1:1000; or 2) rabbit polyclonal anti-whole cell lysate (WCL) antibodies (NR-13819) at 1:5000 or pooled mouse monoclonal anti-LpqH (NR-13822), GroEL2 (NR-13790), PstS1 (NR-13790) antibodies from BEI Resources, NIAID, NIH at 1:500 (4 °C, overnight). This is followed by membrane incubation in HRP-labelled secondary antibodies (goat anti-mouse Ig (1:1000), swine anti-rabbit IgG (1:10,000), and rabbit anti-human IgG (1:5000)) (RT, 1 h). Finally, membranes were incubated in Luminata Forte Western HRP Substrate (Millipore; Bedford, MA, USA) (RT, 1 min) before imaging (CL-Xposure Film; Thermo Fisher Scientific). Membranes were washed thrice in PBS-0.05% Tween-20 between each incubation step.

A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to determine the molecular weight distribution of the protein present in the Liluva samples (6 samples in total, including the raw soaking and cooking water, spray-dried soaking and cooking water, and freeze-dried soaking and cooking water of split yellow peas) as described by Buhl et al. (2019) with modifications [16 (link)]. Invitrogen™ NuPAGE™ 4–12% Bis-Tris precast gels (Bio-Rad, Richmond, CA, USA) were used to evaluate the protein profile in this experiment. A molecular weight marker (10–250 kDa) was applied to estimate the molecular weight of the protein bands. Prior to heat treatment (100 °C for 5 min), 6 samples (concentration of 0.1%) were mixed 3:1 with the NuPAGE™ LDS Sample Buffer (4X) and Sample Reducing Agent. After heating the mixed solution, 8 μL of the molecular markers and 20 μL of the samples were loaded into the gel. Then, 200 V of a constant current and 45 min of running time were set for the electrophoresis, which was conducted in the running buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS). Next, the gel was stained with Commassie blue G-250 staining solution for 1 h. Afterwards, the gel was destained with the destain solution (20% Methanol, 10% Acetic acid) overnight.