Protein assay kit

Cells were rinsed with phosphate-buffered saline (PBS) before being scraped and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail II. After centrifugation at 14,000 rpm for 15 min, the supernatant fractions were harvested as the total cellular protein extracts. The protein concentration was determined using a protein assay kit (Solarbio Life Science, Beijing, China). The total cellular protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes in 20 mM Tris–HCl (pH 8.0), 150 mM glycine, and 20% (v/v) methanol. Membranes were blocked with 5% nonfat dry milk in 1xPBS containing 0.05% Tween-20 (PBS-T) and incubated with antibodies against p-TOPK, TOPK, p-ERK1/2, ERK1/2, p-RSK2, RSK2, p-c-Jun, total c-Jun, PARP, caspase 3, caspase 7, cleaved PARP, cleaved caspase 3, cleaved caspase 7, or β-actin at 4°C, overnight. Blots were washed three times with 1xPBS-T buffer, followed by incubation with the appropriate horseradish peroxidase-linked immunoglobulin G (IgG). The specific proteins in the blots were visualized using an enhanced chemiluminescence detection reagent and the Amersham Imager 600 (GE Healthcare life Science, Pittsburgh, PA, United States).

According to the method used by Baxter et al. [14 (link)], first the milled rice (about 5 g) was defatted with 20 mL hexane and dried under a hood at 25 °C for one day. Then, albumin, globulin, prolamin, and glutelin were sequentially extracted by deionized water, 2% sodium chloride solution, 60% ethanol solution, and 0.0025 mol/L NaOH solution, respectively. All the extractions were repeated three times, and the protein content was measured using bovine serum albumin as standard using a protein assay kit (Solarbio, Shanghai, China).

The total protein from about 0.1g thymus of each piglet was extracted using a RIPA lysis buffer (APPLYGEN, Beijing, China) supplemented with 1 mM phenylmethanesulfonyl fluoride and phosphatase inhibitor, and the concentrations of total proteins were measured using a BCA (bicinchoninic acid) protein assay kit (Solarbio, China). Protein supernatant was separated by 10% SDS-PAGE and transferred into PVDF membranes. After blocking with 5% skimmed milk powder, the membranes were incubated with appropriate primary antibodies overnight at 4°C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. The membranes were washed thrice for 10 min each, incubated with a SuperSignal chemiluminescent substrate (Pierce), and imaged with ChemiDoc XRS+ Imaging System (Bio-Rad). Blots were semi-quantified using the ImageJ software. Primary antibodies for NF-κB p65 (Bioss, 1:1,000) and p-NF-κB p65, p-IκB-α (Bioss, 1:800) and phospho-IκB-α were used in this study.

The protein in anti-VEGFR-LC-PEG-SOR-NP was extracted with the RIPA solution, and the protein concentration was determined by the protein assay kit (Solarbio) and was adjusted to 3 μg/μL. The extracted protein solution (10 μL) was mixed with 10 μL of the loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A constant voltage of 100 V was maintained for the electrophoresis to the bottom of the separation gel. A constant current of 350 mA was transferred onto the nitrocellulose membrane after 2.5 h. After blocking with 5% skim milk powder for l h, l : 1000 diluted rabbit anti-human KDR monoclonal antibody was added, and it was shaken on a shaker overnight and rinsed with a phosphate buffer containing Tween-20 (PBST) three times for 10 min each time. Then, the 1 : 500 diluted goat anti-rabbit IgG-HRP was added, shaken on a shaker for 1 h, and rinsed with PBST three times for 10 min each time. Then, the samples were exposed with electrochemiluminescence (ECL) reagents and scanned with a scanner.

Each bacterial isolate was examined for nitrogen-fixing ability through the acetylene reduction assay (ARA) method [93 (link)]. All isolates were inoculated in a 25 mL flask comprising 10 mL JNFb (Additional file 6) medium and incubated for 3 days at 30 ± 2 °C. Inside air from the tubes is replaced with 5 mL of acetylene through a syringe and the tubes were incubated for 24 h at 30 ± 2 °C [7 ]. The column (DB-1701, Agilent, Santa Clara, USA) temperature was set at 80 °C, whereas flame ionization detector and injector temperature were 110 °C, with carrier gas nitrogen at a flow rate of 35 mL min^− 1. A gas sample from the tube (0.5 mL) was inserted into the GC-17A gas chromatograph (Shimadzu, Kyoto, Japan) and the peak heights were measured and to compute ethylene production in samples. The total protein concentration of each sample tube was estimated by the protein assay kit (Solarbio, Life Sciences, Beijing).