Pageruler prestained protein ladder 26616

Ammonium bicarbonate, iodoacetamide, and dithiothreitol were purchased from Sigma Company (St. Louis, MO, USA). Trypsin (Trypsin Gold, Mass Spectrometry Grade) was purchased from Promega (Madison, WI, USA). HPLC grade formic acid and acetonitrile were obtained from Fisher Scientific International (Hampton, NH, USA). BCA™ Protein Assay Kit was obtained from Pierce (Rockford, IL, USA). PageRuler Prestained Protein Ladder 26616 was purchased from Thermo (Rockford, IL, USA). Ultra-pure water with a resistance of 18.2 MΩ cm⁻¹ was purified using a Milli-Q system (Millipore, Bedford, VA, USA). All other chemicals were analytic grade. Each liter of Tris-HCl contained 6.057 g Tris and was adjusted to pH 9.5. Nylon membrane filters (0.1 μm) were obtained from Whatman (Maidstone, UK).
The standard peptide NPFLFGSNR was synthesized from GL Biochem. Ltd. (Shanghai, China). The purity of peptide was about 98%–99% according to LC analysis performed by the supplier.
Soybean seeds were provided by the Institute of Crop Science Chinese Academy of Agricultural Sciences (Beijing, China). Soybean products were selected from the Ministry of Agriculture Feed Safety and Bio-availability Evaluation Center (Beijing, China).

TFFs were analyzed by Western blot in healthy, OA, and RA synovial fluid. The samples were diluted with PBS (phosphate-buffered saline), and protein concentration was spectroscopically measured by Bradford protein assay. Each sample (30 µg protein) was mixed with reducing sample buffer (RSB) containing 0.5 M dithiothreitol (DTT), 50% glycerin, 0.05% bromphenolblau, 20% β-mercaptoethanol (β-ME) and boiled for 5 minutes. SDS-PAGE and Western blot analyses were performed as described by [56 (link)]. The membrane was incubated with primary antibodies to TFF1 (dilution 1:100), TFF2 (dilution 1:70), and TFF3 (dilution 1:400) at 4°C overnight before applying the secondary antibodies (dilution 1:4000) conjugated to horseradish peroxidase for 2 h and detecting the bands by chemiluminescence using ECL Plus (Amersham Pharmacia, Uppsala, Sweden). Human stomach served as a positive control for all three peptides. The molecular weights of the detected protein bands were estimated referring to a standard protein marker (PageRuler Prestained Protein Ladder #26616; Thermo Fisher Scientific Inc., Waltham, MA, USA) ranging from 10 to 170 kDa. The used antibodies are listed in Table 6. Alpha-1-antitrypsin and actin were used as internal control (primary antibody dilution 1:2000, secondary antibody dilution 1:4000).

The assembly of tubulin (6 μM) was visualized by turbidity measurements in polymerization buffer (50 mM 2-(N-morpholino)ethanesulfonic acid buffer, pH 6.6, containing 100 mM KCl, 1 mM dithioerythritol, 1 mM MgCl₂ and 1 mM ethylene glycol tetraacetic acid) at 37 °C with or without TPPP1 (3 μM) or TPPP3 (3, 9 or 12 μM). The polymerization was induced by the addition of TPPP proteins. The optical density was followed at 350 nm by a Cary 100 spectrophotometer (Varian, Walnut Creek, Australia). In the pelleting experiments, the polymerized samples (100 μL aliquot) were centrifuged (17,000× g, 15 min, 37 °C), and the pellet (P) and supernatant (S) fractions were separated. When indicated, the proteins alone were also prepared and centrifuged under the same conditions. The pellet fractions were resuspended in 100 μL polymerization buffer. The fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MM indicates the molecular weight marker (PageRuler Prestained Protein Ladder 26616, Thermo Scientific, Waltham, MA, USA).

All manipulations were performed at 4°C. After cultivation, cells were collected by centrifugation at 13200 rpm for 5 min and washed twice with 0.9% NaCl. The pellets were resuspended in 0.5 ml of 0.1 M potassium phosphate (pH 7.5), 0.1 mM EDTA, and 0.4 mM dithiothreitol buffer solution with the addition of 0.1 mM phenylmethylsulfonyl fluoride. The obtained suspensions were sonicated and then centrifuged at 13200 rpm for 5 min. The supernatants were decanted and then subjected to enzymatic reactions and 12% SDS-PAGE. PageRuler Prestained Protein Ladder 26616 (Thermo Scientific) was used to evaluate protein molecular mass.
For protein purification, the cells were resuspended in 15 ml of the abovementioned buffer and disrupted with a French press. The cell debris was eliminated by centrifugation at 6000 rpm for 5 min. Then, the supernatant was applied to Ni-NTA Affinity Resin (Clontech, USA). The protein fractions were eluted with an imidazole gradient from 20 mM (in the binding buffer) to 500 mM (in the elution buffer). The fraction containing the purified protein was dissolved in a buffer with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl and 5% glycerol.

All manipulations were performed at 4 °C. Cells were harvested after cultivation by centrifugation at 13,200 rpm for 5 min and washed twice with sterile 0.9% NaCl.
Crude extracts were prepared using xTractor™ Buffer (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s instructions.
The supernatants were decanted and then subjected to 12% SDS-PAGE. Protein molecular mass evaluation was performed by comparing with PageRuler Prestained Protein Ladder 26616 (Thermo Scientific, Waltham, MA, USA). Pure hexahistidine-tagged DSDs were isolated using the Capturem™ His-Tagged Purification Miniprep Kit (Takara Bio, Mountain View, CA, USA).

NDM-1 protein level was determined by SDS-PAGE followed by western blot over the whole-cell lysates of different E. coli stains. Typically, each logarithmic phase culture was lysed by sonication in sonication buffer (50 mM HEPES, pH 7.3, 100 mM NaCl). Bacterial lysates were harvested by centrifugation and normalized according to total protein concentrations, as quantified by bicinchoninic acid assay (Pierce BCA Protein Assay Kit, Thermo Scientific). All the samples were resolved on a 13% SDS-PAGE gel and electrotransferred to a PVDF membrane (Hybond-P, GE Healthcare). A PageRuler Prestained Protein Ladder #26616 (Thermo) was used as a standard marker. Diluted NDM-1 monoclonal antibody (NOVUS Biologicals) and the secondary antibody goat anti-mouse immunoglobulin G (IgG)/alkaline phosphatase (AP) conjugate were applied after performing the standard blotting procedures. The NDM-1 bands were calorimetrically developed with specified ratio of substrates comprising nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) for 15 min. The software ImageJ^{63 (link)} was used to quantify the signal of each of the bands for analysis. The software GraphPad Prism (version 6.2 for Mac, GraphPad Software, La Jolla CA, USA, www.graphpad.com) was used to analyze the resulting plots.