Seeblue plus2 pre stained standard

The yeast doughs and sourdoughs were lyophilized and grounded in a mortar. An aliquot of 2 g dried dough flour was extracted with 150 mM NaCl at ambient temperature to separate the albumin/globulin fraction of the dough flour. The protein solution was also heated at 60 °C for 30 min to inactivate beta-amylases and then the supernatant was dialyzed against water to remove the salt by a membrane cut-off of 3.5 kDa. Followed by lyophilization, the albumin/globulin fraction of dough flour before and after heat treatment were analyzed by SDS-PAGE. Wheat flour as control was treated as described before. The dried samples were mixed with 4× lithium dodecyl sulfate (LDS) sample buffer and 10× sample reducing agent and boiled for 3 min and separated on NuPage Bis-Tris 10% gel with MES (2-(N-morpholino)ethanesulfonic acid) SDS running buffer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Novex Sharp pre-stained protein standard (LC5800) or SeeBlue Plus2 Pre-stained standard (LC5925, Thermo Fisher Scientific) served as molecular markers (myosin 198 kDa, phosphorylase 98 kDa, bovine serum albumin 62 kDa, glutamic dehydrogenase 49 kDa, alcohol dehydrogenase 38 kDa, carbonic anhydrase 28 kDa, myoglobulin 17 kDa, lysozyme 14 kDa, and aprotinin 6 kDa). Running condition was 200 V for 35 min. The gel was stained by Commaasie Brilliant Blue R-250.

The pattern of p24 protein was assessed by Western blot analysis. After protein denaturation, SDS-PAGE was carried out under reducing conditions in a 4–12% NuPage Bis-Tris protein gel (Thermo Fisher Scientific™, Waltham, MA, USA) and SeeBlue Plus2 Prestained Standard (Thermo Fisher Scientific™, Waltham, MA, USA) as molecular weight marker. The proteins were transferred to a PVDF membrane using the iBlot™ 2 system (Thermo Fisher Scientific™, Waltham, MA, USA). After blocking with Tris-Buffered Saline (Merck, Darmstadt, Germany) with 0.1% (w/v) Tween-20 (Merck, Kenilworth, NJ, USA) with 5% (w/v) skim milk (Merck, Kenilworth, NJ, USA) for 1 h, the membrane was incubated overnight with the anti-HIV-1 p24-mouse monoclonal antibody (Abcam, Cambridge, MA, USA). Afterward, the membranes were washed and incubated with the secondary antibody, anti-mouse IgG conjugated with alkaline phosphatase conjugate labeling (Merck, Darmstadt, Germany) for 1 h. The protein detection was performed using NBT/BCIP 1 Step (Thermo Fisher™, Waltham, MA, USA).

The sample solution was mixed 1:1 with Laemmli Sample buffer (Bio-Rad Laboratories, Inc., California, USA) containing 5 % 2-ME. The sample was boiled at 100 °C for 5 min and cooled in the flowing water. The sample and a molecular weight marker (SeeBlue Plus2 Prestained Standard, Thermo Fisher Scientific, CA, USA) were applied at 10 μL/lane to the NuPAGE 10 % Bis-Tris Gel 1.0 mm*15well (Thermo Fisher Scientific, CA, USA) in a running buffer (NuPAGE MES SDS Running Buffer (20X), Thermo Fisher Scientific, CA, USA). The electrophoresis was performed at a constant-voltage (200 voltage) for 30 min using an XCell SureLock Mini-Cell apparatus (Thermo Fisher Scientific, CA, USA) under reducing conditions. The gels were stained with Rapid CBB KANTO (Kanto Chemical Co., Inc., Tokyo, Japan).

The purity of the product and molar mass of protein were assessed
by applying the SDS-PAGE technique using a method adopted from ref (29 (link)) with modifications. Electrophoresis
was performed using the Thermo Scientific apparatus (Thermo Scientific,
Waltham, MA, USA). The used gel was Invitrogen Bolt 4–12% Bis-Tris
Plus (Thermo Scientific, Waltham, MA, USA). The markers of protein
mass were SeeBlue Plus2 Pre-Stained Standard (Thermo Scientific, Waltham,
MA, USA). The gel was stained using the Coomassie Blue method. The
protein solution of about 2 mg/mL was prepared in double-deionized
water. Two additional 10-fold serial dilutions of stock samples were
prepared. The reduced and nonreduced modes were utilized. Samples
were prepared according to a manufacturer (Invitrogen) procedure.
Briefly, protein solution was dispersed in a 2.5 μL load sample
buffer (LDS). Reduction and alkylation were prepared using the sample
reducing agent (10×)—dithiothreitol (DTT) and iodoacetamide
(IAA), respectively. The samples were then heated for 10 min at 70
°C and introduced to the gel. Nonreduced samples were prepared
without the last step (reduction and alkylation). Running buffer was
MES. The electrophoresis process was executed at a voltage of 200
V. After the separation process, the gel was stained for 20 min. The
discoloration was carried out at least for 24 h in double-deionized
water at room temperature.