Bet v 1.0101 was nitrated with tetranitromethane (TNM), diluted in MeOH, at a molar ratio of 30/1, 15/1, 5/1 and 1/1 TNM/tyrosine, leading to the formation of nitro-Bet v 1.0101 [16] (link). Simultaneously, a mock-control (MeOH only) was generated. After (mock-) nitration, the sample was centrifuged through a 10 kDa ultrafiltration tube (Merck Millipore, Cork, Ireland) to remove the reagents, and rinsed twice with fresh phosphate buffer.
10 kda ultrafiltration tube
Manufactured by Merck Group
Sourced in Ireland, United States, Germany
The 10-kDa ultrafiltration tube is a laboratory device designed for the separation and concentration of macromolecules and particles based on their molecular weight. The tube utilizes a semi-permeable membrane with a 10,000 Dalton molecular weight cutoff, which allows smaller molecules to pass through while retaining larger molecules of interest. This equipment is commonly used in various biological and chemical applications that require sample preparation and analyte enrichment.
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Lab products found in correlation
Bradford protein assay kit, by Transgene (1 mentions)
Beyogold his tag purification resin, by Beyotime (1 mentions)
Methanol, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 kda ultrafiltration tube, by Merck Group (1 mentions)
30 kda ultrafiltration tube, by Merck Group (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ultracentrifuge, by Hitachi (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Tmt reagent kit, by Thermo Fisher Scientific (1 mentions)
Sequence grade modified trypsin, by Promega (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Westernbright ecl hrp substrate, by Advansta (1 mentions)
Ni nta beads, by Smart-Lifesciences (1 mentions)
Pet28a vector, by Genewiz (1 mentions)
Superdex 75 increase 10 300 gl, by GE Healthcare (1 mentions)
10 protocols using 10 kda ultrafiltration tube
1
Nitration of Bet v 1.0101 Allergen
2
Purification and Catalytic Activity of NnOMT6 and N323A
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The crude protein of NnOMT6 and N323A obtained was added to 2 ml of pre-equilibrated BeyoGold™ His-tag Purification Resin (Beyotime Biotech, China) and incubated at 4°C for 4 hours. Thereafter, the lysis buffer and elution buffers containing 250 mM imidazole were used to wash and elute the protein, respectively. The purity of recombinant protein was examined by SDS–PAGE analysis (10% gel) and concentrated using a 10-kDa ultrafiltration tube (Merck Millipore). The concentration of purified enzyme was determined with Bradford Protein Assay kit (TransGen Biotech, Beijing, China).
The catalytic activity of purified NnOMT6 and the mutant N323A was further screened using1 –21 as substrates. One hundred microliters of reaction mixture, including 50 mM potassium phosphate buffer (pH 8.0), 200 μM SAM methyl donor, 100 μM substrates, and 50 μg purified protein, was incubated at 37°C for 12 hours. The reactions were analyzed using UPLC–ESI–QTOF–MS/MS.
The catalytic activity of purified NnOMT6 and the mutant N323A was further screened using
3
Nitration of Allergen Proteins
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Beta-lactoglobulin and OVM were nitrated or sham-nitrated as previously described [6 (link),7 (link)]. Briefly, allergens were nitrated using tetranitromethane (TNM) (Sigma, Vienna, Austria) diluted in methanol (Merck, Darmstadt, Germany) for 60 min. Sham-nitration of allergens was done accordingly without adding the nitrating reagent TNM. After sham-treatment or nitration, reaction was stopped by buffer exchange. Samples were centrifuged through a 10 kDa ultrafiltration tube (Merck Millipore, Cork, Ireland) to remove the reagents (residual MeOH and TNM), at 4000 rpm for 10 min. This step was repeated twice with fresh phosphate buffer (10 mM, pH 7.45). Protein concentrations were determined by amino acid analysis and Bradford assays. The degree of nitration was assessed according to a 3-NT standard curve ranging from 5–200 μmol/l by measuring absorbance at 428–650 nm.
4
Synthesis of Gold Nanoparticles Using HSA
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Under optimized conditions, 2 mL of HSA (10 mgmL−1) in ultrapure water was transferred to 2 mL of HAuCl4 (4.375 mM Au) under constant stirring. After 2 min, 0.5 mL NaOH solution (0.5 M) was added to the mixed solution, wrapped in aluminum foil to avoid light, and stirred continuously at room temperature for 12 h. After the reaction was complete, the pH of the solution was adjusted to 7 with hydrochloric acid, and then the bacteria and impurities were filtered out with a 0.22 μm filter. The solution obtained from the reaction was subjected to ultrafiltration with a 10 KDa ultrafiltration tube (Millipore, MWCO: 10 kDa), centrifuged for 15 min, and concentrated to deep brown with a volume of about 1 mL. The purified sample was stocked at 4 °C for subsequent experiments.
5
Citrus Leaf Protein Extraction
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Six citrus leaf midrib samples were selected (three biological replicate samples from the GCK and the others from the GT) in October 2017. The sample was ground in a mortar in the presence of liquid nitrogen, and 10 mL of lysis buffer (8 M urea, 2% SDS, 1× Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland)) was added to each sample power, mixed using a vortex, then sonicated on ice and centrifugated at 13,000× g for 15 min at 4 °C. The supernatant was filtered using a 0.22 μm filter and precipitated with ice-cold acetone at −20 °C overnight. The precipitations were cleaned with 50% ethanol and 50% acetone three times. 200 μg proteins was diluted by buffer (100 mM Tris, pH = 8.0, 8 M urea) to 100 μL, and then the solution was added to 11 μL DTT (1 M) and incubated at 37 °C for 1 h. The treated samples were put in a 10 kDa ultrafiltration tube (Millipore, Billerica, MA, USA) and centrifuged at 12,000× g for 10 min. Then, 100 μL 55 mM iodoacetamide (IAA) was added to block reduce cysteine residues and it was incubated for 20 min in darkness at room temperature.
6
Preparation of Egg White Ultrafiltration Matrixes
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In this study, fresh unfertilized specific-pathogen-free (SPF) eggs that laid by 20- to 22-week-old chickens were purchased from Beijing Merial Vital Laboratory Animal Technology (Beijing, China) and used within 2 days after laying. Preparation of egg white stored at 37, 20, or 4°C was done as described previously (17 (link)). The final prepared egg white was stored at 4°C for up to 5 days. The egg white ultrafiltration matrixes were prepared with egg white stored at 37°C for 5 days. The egg white was centrifuged with a 30-kDa ultrafiltration tube (Millipore), and the filtrate was called 30-kDa ultrafiltration matrix. The 10-kDa ultrafiltration matrix was obtained by centrifuging the 30-kDa ultrafiltration matrix with 10-kDa ultrafiltration tube (Millipore), and the 3-kDa ultrafiltration matrix was obtained by centrifuging the 10-kDa ultrafiltration matrix with 3-kDa ultrafiltration tube (Millipore). This centrifugation was performed at 4°C. Ultrafiltration matrices with different molecular weight cutoffs were stored at –80°C before use.
7
Isolation and Characterization of Extracellular Vesicles
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The above cultures were collected in 50-ml tubes and centrifuged at 800 g for 10 min followed by 5000 g for 20 min to remove excess worm fragments and other large impurities. The supernatant was then passed through a 0.22-μm filter (Millipore, Darmstadt, Germany) to remove larger vesicles and bacteria. Centrifugation was performed using a 10-kDa ultrafiltration tube (Millipore, Darmstadt, Germany) at 5000 g to concentrate the liquid to the appropriate volume. The supernatant was then ultracentrifuged at 4 °C, 120,000 g for 2 h with an ultracentrifuge (Hitachi, Tokyo, Japan). After the supernatant was discarded carefully, sterile PBS (phosphate buffer saline, PH = 7.4) was used to suspend the bottom vesicles, and the protein concentration of the collected Ts-ML-EVs was measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime, Beijing, China). Samples were individually packaged in tubes and stored at − 80 °C for the next steps.
8
Protein Extraction and Tryptic Digestion
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Leaf petiole tissues were ground into powder in liquid nitrogen and were precipitated according to the manufacturer’s protocol using a ProteoExtract Protein Precipitation kit (CalBiochem, Darmstadt, Germany). Two hundred micrograms of protein sample was diluted by buffer (100 mM Tris, pH 8.0, 8 M urea) to 100 μl and then 11 μl of DTT (1 M) was added to the solution, which was incubated at 37°C for 1 h. The treated samples were added into a 10-kDa ultrafiltration tube (Millipore, MA, United States) and centrifuged at 12,000 g for 10 min. Then, 100 μl of 55 mM iodoacetamide (IAA) was added to the ultrafiltration tube and incubated for 20 min protected from light at room temperature. Subsequently, 50 mM triethylammonium bicarbonate (TEAB) was used as exchange buffer. Then, proteins were tryptic digested with sequence-grade modified trypsin (Promega, WI, United States) overnight at 37°C in a 1:50 trypsin-to-protein mass ratio, and the desalted and lyophilized peptide mixture was labeled using chemicals from the TMT reagent kit (Pierce Biotechnology, IL, United States). Samples were then desalted and dried by vacuum centrifugation.
9
Quantification of TFPI2 Protein Levels
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Cell supernatant or lysates derived from C918 and OCM-1A cells were used to examine TFPI2 protein levels. For cell supernatant collection, media of culturing cells were refreshed with FBS-free DMEM. Supernatant and cell lysates were obtained 48 h later. Supernatant enrichment was performed using a 10 kDa ultrafiltration tube (50 ml, Millipore) and centrifuged. Western blotting was done as described previously (38 (link)). Whole cell lysate was separated, transferred onto PVDF membranes, and probed with TFPI2 (Abcam, ab186747, 1:500) and GAPDH (Santa Cruz Biotechnology, sc25778, 1:2,000). Blots of target proteins were detected with WesternBright ECL HRP substrate (R-03031-D2, advansta) and visualized using a C-DiGit Blot Scanner (LI-COR Biosciences).
10
Recombinant Foldon-Fused HR1 Peptides
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HR1SFd, HR1MFd, and HR1LFd peptides were obtained by fusing the T4 bacteriophage fibritin-derived foldon peptide directly to the C terminus of HR1-derived short (HR1S), medium (HR1M) and long (HR1L) peptides, respectively (Fig. 1C ). The genes of HR1SFd, HR1MFd, and HR1LFd were synthesized and cloned into a pET28a vector by Genewiz, Suzhou, China.
pET28a-HR1SFd, pET28a-HR1MFd, and pET28a-HR1LFd plasmids were transformed intoE. coli BL21(DE3), respectively. The cells were incubated at 37°C in an LB medium until the OD600 reached 0.8. The peptides were induced with 0.5 mM isopropyl 1-thio-β-d -galactopyranoside for 6 h at 37°C. Then the cells were harvested, sonicated in PBS with 1% Triton and centrifuged at 1,2000 g for 10 min. The supernatant was loaded into Ni-NTA beads (Smart-Lifesciences, catalog no. SA004100) and washed with 10 mM imidazole in PBS. Peptides were then eluted with elution buffer (250 mM imidazole in PBS). The peak fractions were collected and concentrated with a 10 kDa ultrafiltration tube (Millipore, Germany). The peptides were then analyzed by size exclusion chromatography (Superdex 75 Increase 10/300 GL, GE Healthcare) in the CH3COONa/CH3COOH buffer (pH 6.0).
pET28a-HR1SFd, pET28a-HR1MFd, and pET28a-HR1LFd plasmids were transformed into
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