The collected livers were immediately homogenized at 4 °C in PBS, at pH 7.4 to produced 10% homogenate. The liver homogenate was centrifuged at 5100 g for 15 min, for serum separation. Homogenates were filter through ultra filters (Millipore) and the filtrates were used for determining optical density at 595 nm by using plate reader (BioRad). Uric acid level was measured by Uric Acid Assay Kit (BioAssay Systems) according to standard procedure.46 (link)
Ultrafilter
Manufactured by Merck Group
Sourced in Germany, United States
The Ultrafilter is a piece of laboratory equipment used for the separation and purification of macromolecules, such as proteins, nucleic acids, and other large molecules, from complex mixtures. It operates based on the principle of size-exclusion chromatography, allowing the selective passage of smaller molecules through a semi-permeable membrane while retaining the larger target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Uric acid assay kit, by BioAssay Systems (1 mentions)
Tapi 2, by Merck Group (1 mentions)
Exoquick tctm exosome precipitation solution, by System Biosciences (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ultrafiltration membrane, by Merck Group (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
10 protocols using ultrafilter
1
Liver Homogenate Uric Acid Analysis
2
Exosome Isolation from HepG2 Cells
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HepG2 cells were cultured in 25cm2 flasks. When the confluency reached 60-70%, the cells were washed by PBS twice and counted, and the medium was changed to fresh serum-free DMEM with or without TAPI-2 (Sigma) at the final concentration of 25 μmol/L 18 (link). After 24h, the culture supernatants were collected and concentrated by ultrafilters (Millipore) with molecular weight cut-offs of 10,000. The exosomes were prepared from the concentrated supernatants using the ExoQuick-TCTM Exosome Precipitation Solution (System Biosciences, USA) as described by the manufacturer 7 (link). Meanwhile, the cells were scraped and lysed by cold lysis buffer (Thermo Fisher) with protease inhibitor cocktail (Sigma).
3
Catalytic Production of Hydroxyl Radicals
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PBS (pH = 5.8) solutions of methylene blue (MB) (10 μg/mL) containing Au@FePt nanoprobes (Fe concentration: 100 μg/mL) and different concentrations of H2O2 (0, 2, 4, 8 and 10 mM) were incubated in a 37 °C aqueous bath for 30 min. Then, the obtained solutions were centrifuged at 6000 rpm for 20 min in ultrafilters (Millipore, MWCO = 30 kDa). Afterward, the absorbance changes of MB were measured. To investigate the influence of Fe concentration on the production of ·OH, the absorbance of MB and fluorescence spectra in PBS (pH = 5.8) treated with gradient Fe concentrations (0, 10, 20, 40, 80, 100 μg/mL) and H2O2 (10 mM) were measured.
4
Determining Encapsulation Efficiency of OPC-SDEDDS
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In order to measure encapsulation efficiency (EE), centrifugal ultrafiltration was used to separate the free OPC in the SDEDDS (Fan, Liu, Huang, Li, & Xia, 2014 ). At first, 0.5 g of solid OPC‐SDEDDS was added dispersedly into 9.0 g of deionized water, and note that, dispersion was placed in the upper chamber of centrifuged tube with an ultrafilter (30 kDa, Millipore) and spun at 13,225 g for 30 min at 4°C. The OPC in SDEDDS (total drug) and ultrafiltrate (free drug) was determined by UV‐visible spectrophotometer. EE was calculated from the Equation 3 , and drug loading content was calculated from the Equation 4 . All experiments were verified in triplicate.
where W (total) is the total amount drug in OPC‐SDEDDS, W (free) is the amount of unentrapped drug, and W (excipients) represents the weight of added excipients in the preparation stage.
where W (total) is the total amount drug in OPC‐SDEDDS, W (free) is the amount of unentrapped drug, and W (excipients) represents the weight of added excipients in the preparation stage.
5
Isolation and Analysis of Polybia dimorpha Venom
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Females of Polybia dimorpha were collected at Brasília, Distrito Federal, Brazil, under license in accordance with the Normative Instruction No. 154, from March 2007, of IBAMA (Brazilian Institute of Environment and Renewable Natural Resources, license number 21723–1). Moreover, authorization of the Access and Remittance of the Brazilian genetic patrimony was obtained from the CNPq (license number 010476/2013-0). The wasps were sacrificed after freezing at -20°C. Following species identification (Prof. Fernando B. Noll, UNESP-SP), the venom sacs were dissected and macerated in a 10% acetonitrile in deionized water solution and centrifuged at 5000 × g for 10 min, at 4°C. The supernatant was carefully collected and submitted to a filtration using an ultra-filter (Millipore) with a 3-kDa cut-off for 30 min at 5000 × g. The resultant ultrafiltrate, which was characterized by the presence of low molecular mass compounds, was collected, lyophilized and weighed.
6
Purification of 6xHis-tagged Proteins
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The harvested post-induced cells by centrifugation were resuspended with 40 ml lysis buffer and disrupted using ultra-sonication. Cell slurry was separated into cell-free lysate and cell debris at 10,000 × g for 30 min at 4°C. The resulted cell-free lysate was gently mixed with 1 ml 50% Ni-NTA slurry at 4°C for 1 h. The mixture was loaded onto a column and washed with 100 to 200 ml washing buffer until no protein eluted in the flow-through. The 6 × His tagged proteins bound to Ni-NTA resin were eluted with 5 ml elution buffer. The eluted protein fraction was concentrated using Millipore ultrafilter with 30-kDa molecular weight cutoff (2,000 × g, 30 min, 4°C) and loaded onto a pre-equilibrated PD-10 column for buffer exchange using desalting buffer. All protein fractions were monitored using Bradford Protein Assay kit. The aliquots of collected fractions were flash frozen by liquid nitrogen and stored at −80°C for later use.
7
Yeast Surface Display Protocol
8
Peptide Fractionation by Ultrafiltration
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The hydrolysate sample was fractionated using ultrafiltration membrane (Millipore, Darmstadt, Germany) according to the instructions in the manual (Amicon® Ultra filter) with MW cut-offs of 3, 10, and 30 kDa. The permeates or retentates were collected as >30 kDa, <30 kDa, 10–30 kDa, <10 kDa, 3–10 kDa, and <3 kDa, respectively. All permeates were stored at −20 °C for further analysis.
9
Isolation and Characterization of UC-MSCs
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The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The use of the human umbilical cord tissue from a healthy donor who gave birth and signed informed consent in Renmin Hospital was supported by the Institutional Ethics Review Board of Renmin Hospital of Wuhan University (Permit Number: WDRY2019-G001). UC-MSCs were isolated, cultured and identified as described in our previous study (19 (link)). Briefly, the umbilical cord was cut into small pieces and cultured in serum-free medium (UltraCULTURE, Walkersville Lonza, MD) supplemented with serum alternatives (Life Science, France) and L-Glutamine at 37 °C in a humidified atmosphere containing 5% CO2. The fifth generations of cells were used in this animal experiment. UC-MSCs were characterized for surface marker expression and differentiation potential of adipogenesis, osteogenesis, and chondrogenesis as described previously (19 (link)). For the production of UC-MSCs condition medium (UC-MSCs-CM), the supernatant of UC-MSCs was collected and concentrated 25-fold using ultrafilter (Millipore, Bedford, MA), when the confluence reached about 80%.
10
Purification of Apple Juice Polyphenol Oxidase
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After thermal treatment, apple juice was collected and fractionated with (NH4)2SO4 at 25% saturation to remove impurities and 80% saturation to precipitate the protein for 1 h with PPO activity. The sample was then centrifuged (Eppendorf Centrifuge 5804 R, Germany) at 10,000 rpm for 20 min at 4°C. The precipitate was resuspended in 0.5 mol/L Tris–HCl buffer (pH 7.0). The sample was dialysed against 0.5 mol/L Tris–HCl buffer (pH 7.0) for 24 h, and the buffer was changed after 1 h during dialysis. The supernatant containing proteins was concentrated using an ultrafilter (Millipore Co., Bedford, MA, USA) and purified using the methods reported in our previous study (Cheng et al., 2014 (link)) by using DEAE Sepharose Fast Flow and Sephacryl S-200 columns (2.6 × 30 cm2) preequilibrated with 0.05 M Tris–HCl buffer (pH 7.0) respectively.
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