The liver was rapidly rinsed in 250 mmol/L ice-cold sucrose, blotted on absorbing paper, and weighted. Microsomes were isolated according to the procedure of Henne and Soling [18 (link)], modified as follows. Livers were homogenized (10% w/v) in (mmol/L): 250 sucrose, 20 K-HEPES (pH 7.2) in a Thomas C potter by 4 passes with a tight-fitting Teflon pestle. Homogenate was sequentially centrifuged in a JA-20 Beckman rotor at 2,500g x 10 min to remove nuclei and cellular debris, and at 10,000g x 10 min to remove mitochondria. The post-mitochondria supernatant was sedimented at 80,000g x 45 min in a Ti 90 Beckman rotor. The glycogen pellet was discarded. The microsomal pellet was washed once with cytosol-like, Mg2+ free medium (incubation medium) previously described, and centrifuged at 80,000g x 45 min. The final pellet was resuspended in the described incubation medium at a concentration of 15 mg/ml, and stored in liquid nitrogen until used.
Beckman ja 20 rotor
Manufactured by Beckman Coulter
Sourced in United States
The Beckman JA-20 rotor is a high-performance centrifuge rotor designed for a wide range of laboratory applications. It is capable of reaching a maximum speed of 20,000 rpm and can generate a maximum relative centrifugal force (RCF) of 48,400 xg. The rotor is compatible with a variety of sample tubes and bottles, allowing for efficient separation and purification of biological samples.
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5 protocols using beckman ja 20 rotor
1
Isolation of Liver Microsomes
2
Purification of GST-tagged Proteins
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Pellets were suspended in GST-lysis buffer (15 mL for 1 L culture). Samples were sonicated 3 times for 30 s/each on ice and were pelleted down at 13,200 rpm for 30 min at 4 °C using a JA 20 Beckman rotor or at 40,000 rpm for 45 min at 4 °C using a 55.2 Ti Beckman rotor. A total of 1 mL of glutathione-sepharose beads (Amersham), previously washed 3 times with GST-lysis buffer, was added to the supernatant and samples were incubated 1–2 h at 4 °C while rocking. Beads were then washed 3 times (with 5 min of incubation at 4 °C each) in the GST lysis solution. GST-proteins were resuspendend 50% slurry in the GST-lysis solution. The quantification was achieved in an SDS PAGE gel using a titration curve with BSA.
3
Phage Particle Purification and Visualization
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A preparation of phage particles was centrifuged at 41,200× g for 120 min at 4 °C in a Beckman JA-20 rotor (Beckman Coulter, Fullerton, CA, USA) and suspended at about 1012 PFU/mL in SM buffer; 10 μL of the concentrated phage suspension were let to adsorb on a carbon-coated matrix and then stained with 2% uranyl acetate for 15 s. The grid was subsequently washed twice with ddH2O, air dried and observed by a FEI Tecnai 12 (FEI, Eindhoven, The Netherlands) TEM fitted with an Osis Morada 2X4 K CCD camera (Olympus, Shinjuku, Tokyo, Japan).
4
Efficient VLP Production in HEK293 Cells
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VLPs were expressed in HEK293 cells by transient transfection and concentrated by ultracentrifugation. Twenty-four hours prior to transfection cells were seeded at a density of 4×105 cells ml−1 in 175 cm2 T flasks and incubated overnight (ON) at 37 °C and 5% CO2. The transfection was performed using 5 μg of DNA per 106 cells and PEI/DNA polyplexes were formed by adding PEI to plasmid DNA (in a 1.5:1 ratio) diluted in DMEM. Complexes were incubated for 15 min at room temperature (RT) and added to the culture. Four hours post-transfection (hpt), cell supernatant was removed and replaced by fresh complete medium containing valproic acid (1.68 mM) and caffeine (2.5 mM) (Sigma-Aldrich).
Seventy-two hpt cell supernatant containing VLPs was harvested and clarified by centrifugation at 4000 g for 20 min. The clarified supernatant was layered over a 30% sucrose cushion and centrifuged at 60,000 g for 5 h at 4 °C (Beckman JA-20 rotor, Beckman Avanti J-E, Beckman Coulter). VLP pellet was resuspended in PBS using 1% of the original volume and stored at 4 °C.
Seventy-two hpt cell supernatant containing VLPs was harvested and clarified by centrifugation at 4000 g for 20 min. The clarified supernatant was layered over a 30% sucrose cushion and centrifuged at 60,000 g for 5 h at 4 °C (Beckman JA-20 rotor, Beckman Avanti J-E, Beckman Coulter). VLP pellet was resuspended in PBS using 1% of the original volume and stored at 4 °C.
5
Pseudotyped Lentivirus Production and Transduction
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An established protocol for pseudotyped lentivirus production was followed (Crawford et al. 2020 (link)). Briefly, HEK293T cells were co-transfected using PEI with the plasmids HDM-Hgpm2, HDM-tat1b, pRC-CMV-Rev1b, and pHAGE2-CMV-ZsGreen-W, and a plasmid encoding the envelope protein, i.e., HDM-IDTSpike-fix, for wt S protein or pLV-S-RVG for the fusion protein pseudotype. As a negative control, a lentiviral stock without any envelope protein was constructed. Forty-eight hours post transfection, supernatants were collected and clarified at 4000 g for 20 min, and then concentrated by ultracentrifugation at 60,000 g for 5 h at 4 °C (Beckman JA-20 rotor, Beckman Avanti J-E, Beckman Coulter). Pellets were resuspended in DMEM with 3% of the original volume and stored at −70 °C.
HEK293 cells expressing ACE2 were used to analyze S-RVG ability to interact with ACE2 receptor and mediate lentiviral transduction. For this, HEK293-ACE2 or wt HEK293 cells were plated on 96-well plates at 3.5×104 cells ml−1 24 h prior to the transduction experiment. Then, culture supernatants were removed and concentrated lentivirus samples diluted 1:2 in DMEM complete medium were added over the cells by triplicate. Seventy-two hours later, ZsGreen expression on transduced cells was measured by flow cytometry with a GUAVA EasyCyte cytometer using Guava Express Plus Software.
HEK293 cells expressing ACE2 were used to analyze S-RVG ability to interact with ACE2 receptor and mediate lentiviral transduction. For this, HEK293-ACE2 or wt HEK293 cells were plated on 96-well plates at 3.5×104 cells ml−1 24 h prior to the transduction experiment. Then, culture supernatants were removed and concentrated lentivirus samples diluted 1:2 in DMEM complete medium were added over the cells by triplicate. Seventy-two hours later, ZsGreen expression on transduced cells was measured by flow cytometry with a GUAVA EasyCyte cytometer using Guava Express Plus Software.
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