The purification of wt-EV71 virions was performed by using a previously reported protocol with modifications [21] (link). In brief, RD cells in five T175 cell culture flasks were infected with EV71 (Anhui1 strain) at an MOI of 0.1 and cultured in 10% FBS. When 90% of the cells exhibited CPE, the supernatant was collected and concentrated by filtration through a 100 kDa-cutoff centrifugal filter (Millipore). The concentrated virus was mixed with 1.4 g/ml CsCl at a volume ration of 1∶4 and loaded on the middle of a CsCl gradient (1.1 g/ml, 1.2 g/ml, 1.3 g/ml, 1.4 g/ml, and 1.5 g/ml, discontinuously) followed by ultracentrifugation at 41,000 rpm for 10 h at 4°C in a Beckman SW41Ti rotor. After being dialyzed with PBS, the purified EV71 virus was quantified and stored at -80°C. The RNA copies of purified EV71 virus was quantified as previously described.
100 kda cutoff centrifugal filter
Manufactured by Merck Group
Sourced in United States
The 100 kDa-cutoff centrifugal filter is a laboratory equipment designed for sample concentration and buffer exchange. It utilizes centrifugal force to separate molecules based on their molecular weight, retaining molecules larger than the 100 kDa membrane cutoff while allowing smaller molecules to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Sw41ti rotor, by Beckman Coulter (1 mentions)
P70at rotor, by Hitachi (1 mentions)
0.45 μm membrane, by Merck Group (1 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
0.22 μm membrane, by Merck Group (1 mentions)
Dpni restriction enzyme, by New England Biolabs (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Lauryl maltose neopentylglycol lmng, by Anatrace (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
4 protocols using 100 kda cutoff centrifugal filter
1
Purification of Enterovirus 71 Virions
2
Isolation and Characterization of R. equi-Derived Extracellular Vesicles
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EVs produced by 103+ (103+-EVs) or 103− (103−-EVs) were Isolation by ultracentrifugation. Briefly, sub-cultured R. equi were inoculated to BHI and grown at 37 °C with vigorous shaking to OD600 = 1.0. R. equi were pelleted at 12,000× g for 20 min, the supernatant was again at 16,000× g for 20 min to remove remaining cells. The supernatant was filtered through a 0.45 μm membrane (Millipore, Burlington, MA, USA), and concentrated 20-fold with 100-kDa cut-off centrifugal filter (Millipore, Burlington, MA, USA). The retentate was again filtered through a 0.22 μm membrane (Millipore, Burlington, MA, USA). The resulting filtrate was subjected to ultracentrifugation at 150,000× g for 3 h at 4 °C using a P70AT rotor (Hitachi, Tokyo, Japan). The pellet obtained was re-suspended in phosphate-buffered saline (PBS) and again subjected to ultracentrifugation at 150,000× g for 3 h at 4 °C. The precipitate was re-suspended in PBS. The protein concentrations of R. equi-EVs were quantified using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc, Waltham, MA, USA). Finally, R. equi-EVs were stored at −80 °C until further characterization. Additionally, 10 μL of purified R. equi-EVs was grown in BHI agar plates to confirm that all R. equi cells were eliminated.
3
Eliminating Bacterial DNA Contamination
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The previously established Dpn I digestion combined with size-exclusion ultrafiltration method was used to remove the potential bacterial DNA contamination with slight modification (28 ). By virtue of selective cleavage of G(m6dA)TC sites in DNA by Dpn I restriction enzyme (39 (link),40 (link)), bacterial DNA can be cut into short fragments since adenines in bacterial DNA are heavily methylated to m6dA. However, GATC sites in DNA cannot be cut by Dpn I. Thus, the DNA of mammals remains in large-size after digestion by Dpn I since m6dA generally has very low abundance in genomic DNA of mammals. The resulting small DNA fragments from bacterial after Dpn I digestion can be removed from large-size genomic DNA of mammals using size-exclusion ultrafiltration. Briefly, a 50-μl mixture containing 10 μg of genomic DNA, 5 μl 10× cutsmart buffer (New England Biolabs), 80 units of Dpn I restriction enzyme (New England Biolabs) was incubated at 37°C for 1 h. Then the reaction products were transferred to a 100 kDa cut-off centrifugal filter (Millipore) and centrifuged at 8000 g for 3 min at 4°C. The centrifugal filter was washed three times with 500 μl of cold water for each time. The DNA retained on the filter was then collected and dissolved in water.
4
Membrane Protein Purification for Cryo-EM
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The cell pellets were resuspended in buffer A [20 mM Hepes (pH 7.5), 150 mM NaCl, and 10% glycerol] and disrupted by passing through a high-pressure homogenizer at 1200 bar. Following centrifugation (27,000g, 15 min, 4°C), the membrane-containing supernatant was applied to ultracentrifugation at 150,000g for 1.5 hours. The collected membrane fractions were resuspended in buffer A and stirred at 4°C for 1.5 hours in the presence of 1% (w/v) Lauryl maltose neopentyl glycol (LMNG) (Anatrace), and then ultracentrifuged at 40,000g for 40 min. The supernatant was incubated with anti-flag beads (GenScript) at 4°C for 1.5 hours. The beads were washed by buffer A supplemented with 0.005% (w/v) LMNG. The protein was eluted with buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.06% (w/v) digitonin, and flag peptides (0.2 mg ml−1). The elution was concentrated to a volume of ~500 μl using a 100-kDa cutoff centrifugal filter (Millipore) and loaded to a size exclusion chromatography column (Superose 6 Increase 10/300 GL, GE Healthcare) pre-equilibrated with 20 mM Hepes (pH 7.5), 150 mM NaCl, and 0.06% (w/v) digitonin. The peak fractions were pooled and concentrated to 5.5 mg ml−1 for cryo-EM. All mutants were purified following a similar protocol.
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