Spike S1 (40591-V08H; Sino Biological) was conjugated to lentivirus (Cellomics Technology LLC) to create a SARS-CoV-2 mimic. His-tagged Spike S1 was linked to Ni nitrilotriacetate (Ni-NTA) through the chemical interaction. NTA with mercapto group (N-[Nα,Nα-Bis(carboxymethyl)-L-lysine]-12-mercaptododecanamide) was first reacted with 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) to give NTA-SMCC and then was added to the lentivirus. The NTA groups were conjugated to the lentivirus through the −NH2 group on lentivirus and N-hydroxysuccinimide ester on NTA-SMCC. The free NTA-SMCC was removed by centrifugation using an ultrafiltration tube (100 kDa MWCO; Millipore) to give SARS-CoV-2 mimicking virus (Spike S1-lentivirus). The successfully conjugated Spike S1 on lentivirus was confirmed using TEM. Briefly, SARS-CoV-2 mimics were incubated with anti-Spike S1 antibodies overnight at 4°C. Free antibodies were removed using an ultrafiltration tube (100 kDa MWCO; Millipore) and washed with PBS three times. Spike S1 on the SARS-CoV-2 mimics was labeled with immunogold (10 nm) antibodies and negatively stained for TEM visualization. The conjugation efficiency of Spike S1 on lentivirus was determined using ELISA (Sino Biological SARS-COV-2 SPIKE ELISA KIT, Sino Biological) according to manufacturer’s protocol.
Ultrafiltration tube
Manufactured by Merck Group
Sourced in United States, Germany
The Ultrafiltration tube is a laboratory equipment designed for the separation and concentration of macromolecules, such as proteins, from complex solutions. It utilizes a semi-permeable membrane to selectively allow the passage of smaller molecules while retaining the desired macromolecules. This process is known as ultrafiltration, which is a widely used technique in various scientific and industrial applications.
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Lab products found in correlation
2 d quant, by GE Healthcare (4 mentions)
Lentivirus, by Merck Group (2 mentions)
Lentivirus, by Sino Biological (2 mentions)
Sars cov 2 spike elisa kit, by Sino Biological (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Bradford protein assay kit, by Beyotime (2 mentions)
0.22 μm filter, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ni nta sefinose his bind column, by Bio Basic (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Modified bca assay, by Thermo Fisher Scientific (2 mentions)
Transmission electron microscope, by Philips (2 mentions)
Ce tofms, by Agilent Technologies (1 mentions)
Ultrasonic cell crusher, by Ningbo Scientz Biotechnology (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression media, by Thermo Fisher Scientific (1 mentions)
Mouse anti his tag antibody, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose column, by Qiagen (1 mentions)
Sod assay kit, by Nanjing Jiancheng (1 mentions)
105 protocols using ultrafiltration tube
1
SARS-CoV-2 Spike Protein Lentivirus Mimic
2
SARS-CoV-2 Spike Protein Lentivirus Mimic
3
Robust Metabolite Extraction and Quantification
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For each sample, metabolites were extracted from the frozen tissue powder by 1 ml methanol containing 20 µM each of L-Methionine sulfone, 2-morpholinoethanesulfonic acid, monohydrate, and sodium d-camphor-10-sulfonic acid. 500 µl of the lysate was transferred to an Eppendorf tube containing 500 µl chloroform and 200 µl of Milli-Q water. After 30 s of vortexing and 15 min of centrifugation at 4°C, 300 µl of the aqueous phase was concentrated and dried to complete dryness in an ultrafiltration tube (Millipore) followed by speed vacuum for 3 hr at 35°C. The dried samples were mixed with 100 µl of Milli-Q water containing 100 µM each of 3-aminopyrrolidine and trimesate, and filtered with an ultrafiltration tube (Millipore) at 9100 × g for 2 hr at 4°C immediately before 7 µl was used in a CE-TOF-MS (Agilent Technologies) to detect cationic metabolites and anionic metabolites. The instrumentation and measurement conditions used for CE-TOF-MS were according to Sugimoto et al., 2012 (link).
The in-house software MasterHands was used for peak detection, time alignment, and peak area integration. The intensities of each metabolite in the samples were calculated based on the comparison of peak area normalized by internal standards added to the sample as well as external standards. Metabolites detected in less than half of the samples were excluded from the following analysis.
The in-house software MasterHands was used for peak detection, time alignment, and peak area integration. The intensities of each metabolite in the samples were calculated based on the comparison of peak area normalized by internal standards added to the sample as well as external standards. Metabolites detected in less than half of the samples were excluded from the following analysis.
4
Synthesis of BPBBT-CL-HSA Nanoparticles
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BPBBT (1 mg/mL) was dissolved in dichloromethane. HSA (2 mg/mL) was diluted with phosphate-buffered saline (PBS). The HSA solution was mixed with BPBBT solution and the mixture was homogenized for 2 min with a homogenizer (Jinxin, Shanghai, China) to form a crude emulsion. The emulsion was transferred and sonicated with ultrasonic cell crusher (Scientz, China) for 5 min under ice bath. The organic solvent was rapidly removed at reduced pressure for 10 min. The BPBBT-HSA NPs were obtained by filtration (0.22 μm, Millipore, USA) and washed three times using an ultrafiltration tube (MWCO 100 kDa, Merck, USA). For the preparation of BPBBT-CL-HSA NPs, the prepared BPBBT-HSA NPs were cross-linked by the addition of 8% glutaraldehyde solution (1.175 μL/mg HSA) followed by stirring for 24 h at room temperature [35 (link),36 (link),37 (link)]. The BPBBT-CL-HSA NPs solution was washed three times using an ultrafiltration tube (MWCO 100 kDa, Merck, USA).
5
Urine Extracellular Vesicle Isolation Protocol
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A total of Fifty ml of mid-morning urine sample were obtained from each participant. Collected specimens underwent centrifugation at 1500 g for 10 minutes at a temperature of 4 °C and followed by 10,000 g for 30 minutes to remove the cells and debris. The urine sample was further concentrated to 10 ml using ultrafiltration tubes (Millipore, 10kd). a total of 50 mL of phosphate buffer solution (1xPBS) was utilized to wash the 10/35-nm qEV10 size-exclusion chromatography columns (SECs, H-wayen Biotechnologies, Shang Hai, China). Then, concentrated samples were dropped to SECs. The beginning 10 ml of liquids was discarded, and then 20 ml liquids were saved. Lastly, saved 20 ml liquids were further concentrated to 1 ml using ultrafiltration tubes (Millipore, 10kd) at a speed of 5000 g for 15 ~ 28 minutes and stored at a temperature of -80 °C until utilization.
6
Grub Polypeptide Extraction Protocol
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The extraction of grub polypeptide was based on the previous study (Li et al. 2019 ). Briefly, drying grubs were ground into powder, then the water was mixed with the powder in a ratio of 25:1. 6000 U of papain per liter was added to the mixture for 2 h at 55°C to enzymatic hydrolyze it. The extraction rate was 20–30%. Centrifuging the extraction at 4000 rpm for 20 min and getting the crude extracts. Filtering the crude extracts with ultrafiltration tube (Millipore Corporation, USA) and obtaining the grubs polypeptide extracts. Analysis of the content of polypeptide extracts with biuret assay.
7
Bacterial Cell Protein Extraction Protocol
8
ABTS Cation Radical Scavenging Kinetics
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ABTS cation radicals (ABTS+) were obtained from the oxidation of ABTS (10 mmol/L) with the laccase (50 U/L) in 50 μmol/L phosphate–citrate buffer (pH 3) at 40 °C for 2 h. Then, the reaction mixtures were centrifuged in an ultrafiltration tube (molecular weight cutoff 10 kDa; Millipore, Bedford, MA, USA) at 9000× g for 10 min). Enzymes and polymerized substances were removed. The oxidized products were incubated with buffer (30 mM, pH 2~7) and ionic liquids (50 mM) at 25 °C for 6 min. The concentration of each product was monitored by the spectrophotometer.
The first-order kinetics model was assumed to describe the time-dependent scavenging of ABTS+, which generally showed an exponential decrease in the plot with linear axes (Figure S6 ). The variation of ABTS concentration, Ln(Ao)-Ln(At), versus time (t) gave straight lines (Equation (5)). Half-life (t1/2) was a constant value and could be estimated from the rate constant (K) according to Equation (6).
The first-order kinetics model was assumed to describe the time-dependent scavenging of ABTS+, which generally showed an exponential decrease in the plot with linear axes (
9
Synthesis and Hydrolysis of mPEG-poly(1/3)
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mPEG−poly(1 /3 ) was dissolved in dimethyl sulfoxide (DMSO) and slowly added into 0 °C PBS. The mixture solution was ultrafiltrated by an ultrafiltration tube (molecular weight cut-off (MWCO) = 10,000 Da; Millipore Co., Billerica, MA, USA). Finally, the concentration of mPEG−poly(1 /3 ) solution was kept at 1.0 mg mL−1.
To investigate the hydrolysis of mPEG−poly(1 /3 ), PUM was shaken at 80 rpm in a 37 °C incubator, 3.0 mL of the liquid was pipetted at different time points, and the liquid was lyophilized. Finally, the lyophilized solids were dissolved in DMF, and Mns were monitored by GPC.
To investigate the hydrolysis of mPEG−poly(
10
Ancient DNA Extraction from Bone and Tooth
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Ancient DNA extractions were performed in a dedicated ancient DNA laboratory at the China University of Geosciences (Wuhan), physically separated from post-PCR facilities. Approximately 200 mg of bone or tooth powder of each sample was digested in extraction buffer which contained 4.5 mL of EDTA (0.5 M, pH = 8) and 0.06 mL of Proteinase K (20 mg/mL), followed by incubation for 16 h in a rotating hybridization oven at 37 °C. After centrifugation at 7000 rpm for 10 min, the supernatant was transferred into an ultrafiltration tube (Millipore, Darmstadt, Germany) and condensed to 100 μL. Finally, DNA was purified and eluted in 80 μL EB buffer using the MinElute PCR Purification Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.
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