Gp120 was resolved by SDS-PAGE, and the monomer excised and washed as described above. Reduction was carried out by addition of 10 mM Dithiothreitol in 100 mM ammonium bicarbonate and incubation at 65°C for 30 mins. Alkylation was subsequently performed by addition of 50 mM Iodoacetamide in 100 mM ammonium bicarbonate, with incubation at room temperature for 50 mins. Trypsin (Promega) was then added at a concentration of 12.5 μg ml−1 and incubated at 37°C for 16 hours. Glycopeptides were eluted from the gel with alternate washes of water and acetonitrile, filtered through a 0.45 μM membrane (Whatman), and dried in a SpeedVac concentrator. After resuspension in 0.1% TFA, the glycopeptide pool was fractionated by reverse phase-HPLC using a Jupiter C18 5 μm 250 × 4.5 mm column (300 Å pore size) (Phenomenex) and a Dionex U3000 LC system. The following gradient was run at a flow rate of 1 ml min−1, with fractions being collected every minute for 90 minutes: time = 0 min (t=0): 95% A, 5% B; t= 5: 95% A, 5% B; t=90: 10% A, 90% B; t=95: 10% A, 90% B; t=97: 95% A, 5% B; t= 120: 95% A, 5% B, where solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was acetonitrile. UV absorbance was detected at 214 and 280 nm.
0.45 μm membrane
Manufactured by Cytiva
Sourced in United Kingdom
The 0.45 μm membrane is a laboratory filtration device designed to remove particulates and microorganisms from liquid samples. It is a porous membrane with a nominal pore size of 0.45 micrometers, which can efficiently filter a wide range of aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Jupiter c18 5 μm 250 4.5 mm column, by Phenomenex (2 mentions)
U3000 lc system, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Softmax pro 5, by Molecular Devices (1 mentions)
Icp ms, by Agilent Technologies (1 mentions)
Ms excel, by OriginLab (1 mentions)
50 ml plastic centrifuge tubes, by Corning (1 mentions)
Originpro 8, by OriginLab (1 mentions)
7 protocols using 0.45 μm membrane
1
Glycopeptide Enrichment and Fractionation
2
Glycopeptide Enrichment and Fractionation
3
Methanolic Extraction of Homogenized Samples
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5 g of each of homogenised sample were extracted with 20 mL of methanol at ambient temperature for 30 min. The supernatant was collected, filtrated through a 0.45 μm membrane (Whatman International Ltd., Maidstone, UK) and diluted up to 25 mL in a volumetric flask with methanol.
4
Quantitative Adipocyte Lipid Assay
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The mature adipocytes were fixed in 4% formalin for 20 min, and then washed with isopropyl alcohol. The fixed cells were then stained with Oil Red O solution for 15 min. Oil Red O solution was prepared by dissolving 0.25 mg of Oil Red O powder in 50 mL of 60% isopropyl alcohol, followed by filtering through a 0.45-μm membrane (Whatman, Maidstone, UK). After staining, the cells were washed twice with PBS. Thereafter, the Oil Red O stain was eluted with isopropyl alcohol, and the absorbance was measured at 515 nm (for 3T3-L1) and 495 nm (for human ASCs) using a spectrophotometer (Softmax pro 5; Molecular Devices, CA, USA).
5
Evaluating Herbicide Effects on Callus Biomass
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Ten callus pieces with initial biomass of 5–6 mg were placed on solid BCGM in a 90-mm Petri dish. In the imazapic experiment, a water–imazapic solution (20 μl) consisting of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM imazapic was passed through a 0.45-μm membrane (Whatman) onto each callus piece. In the glyphosate experiment, glyphosate was embedded in BCGM at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM. Dishes were kept in the dark at 25°C for 4 weeks in the imazapic experiment and for 8 weeks in the glyphosate experiment. Then the calluses from each dish were weighed to calculate biomass accumulation. The callus was immediately frozen in liquid nitrogen and stored at -80°C. Free amino acid content was analyzed in the calluses of all treatments (Hacham et al., 2016 (link)).
In the glyphosate experiment, shikimic acid accumulation (according to Zelaya et al., 2011 (link)) in the callus was analyzed. Both experiments were conducted with eight replicates (Petri dishes) per treatment.
In the glyphosate experiment, shikimic acid accumulation (according to Zelaya et al., 2011 (link)) in the callus was analyzed. Both experiments were conducted with eight replicates (Petri dishes) per treatment.
6
Water Quality Assessment Protocol
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Water samples were filtered aseptically through a 0.45 μm membrane (Whatman, UK) and placed on Membrane Lauryl Sulphate agar for total coliforms and E. coli or Nutrient Agar for total aerobic bacterial count. Appropriate volumes (1, 10 and 100 mL) of the samples were aseptically filtered in duplicate using a vacuum pump attached to the water filtration system and results are expressed as CFU/100 mL. Standard Membrane Lauryl Sulphate total coliforms cultures were incubated aerobically at 35 °C for 24–48 h and for E. coli incubated aerobically at 35 °C for 4 h, then at 44 °C for 44 h while nutrient agar plates were incubated at 37 °C for 24–48 h. The numbers of colony-forming units (CFU) were calculated and numbers were expressed as CFU/100 mL [14 (link)].
7
Uranium Adsorption on Hydroxyapatite
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All adsorption experiments were conducted in duplicates, including blanks and calibration controls. Briefly, 20 mg of HA was weighed into 50 mL plastic centrifuge tubes (Corning, Corning, NY, USA) with 30 mL U solution, and the pH of the suspension was adjusted to 3.0. The tubes were then shaken for 6 h to achieve equilibrium. Then, the tubes were centrifuged, and the supernatants were filtered through a 0.45 μm membrane (Whatman, Little Chalfont, Buckinghamshire, UK) for analysis of U concentration with an ICP-MS (Varian Inc., Palo Alto, CA, USA). The pH at the beginning and end of adsorption experiment was measured by a pH meter (Oakton, Vernon Hills, IL, USA).
U adsorption on the HA was calculated from the difference in concentrations before and after the adsorption. MS-Excel and OriginPro 8.0 (OriginLab, Wellesley Hills, MA, USA) were used for data processing.
U adsorption on the HA was calculated from the difference in concentrations before and after the adsorption. MS-Excel and OriginPro 8.0 (OriginLab, Wellesley Hills, MA, USA) were used for data processing.
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