The viruses in the allantoic fluid were first inactivated using 0.05% BPL as described previously2 . To concentrate the viruses, allantoic fluids were clarified by centrifugation at 3441 × g at 4 °C for 30 min using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). Clarified allantoic fluids were laid on top of a 20% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4). Ultracentrifugation in a Beckman L7-65 ultracentrifuge at 112,499 × g for 2 hours at 4 °C using a Beckman SW28 rotor (Beckman Coulter) was performed to pellet the viruses through the sucrose cushion while soluble egg proteins were removed. The virus pellets were resuspended in PBS (pH 7.4). The total protein content was determined using the bicinchoninic acid assay (Thermo Fisher Scientific).
Beckman sw28 rotor
Manufactured by Beckman Coulter
Sourced in United States
The Beckman SW28 rotor is a high-performance centrifuge rotor designed for the Beckman Coulter ultracentrifuge platform. It is capable of achieving a maximum speed of 28,000 RPM and can generate a maximum relative centrifugal force (RCF) of 149,000 x g. The SW28 rotor is commonly used for a variety of applications, including the separation and purification of biological macromolecules, such as proteins, nucleic acids, and organelles.
Automatically generated - may contain errors
Lab products found in correlation
Sorvall legend rt plus refrigerated benchtop centrifuge, by Thermo Fisher Scientific (4 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (3 mentions)
L7 65, by Beckman Coulter (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Millex gv, by Merck Group (2 mentions)
Dnase 1, by Evrogen (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Beckman sw41 rotor, by Beckman Coulter (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pmd2 g, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Cycloheximide (chx), by AppliChem (1 mentions)
Phenol chloroform isoamyl alcohol, by AppliChem (1 mentions)
Ht7700, by Hitachi (1 mentions)
Prism 5, by GraphPad (1 mentions)
Beckman sw 55 rotor, by Beckman Coulter (1 mentions)
13 protocols using beckman sw28 rotor
1
Virus Concentration and Purification from Allantoic Fluid
2
Influenza Virus Purification and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Influenza viruses were grown in 10-day old embryonated chicken eggs at 37 °C for two days, and were then cooled at 4 °C overnight. Allantoic fluids were collected and clarified by low speed centrifugation. Viruses in the clarified allantoic fluids were inactivated with 0.03% methanol-free formaldehyde for 48 h at 4 °C with rocking. Viruses were then pelleted through a 30% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) by centrifugation in a Beckman L7-65 ultracentrifuge at 25,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Pellets were collected in PBS (pH 7.4), and protein content was quantified using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).
3
Lentiviral Vector Production Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
High-titer replication-defective lentiviral vectors were produced and concentrated as previously described [27 (link),28 (link)]. Briefly, 293 T human embryonic kidney cells were transfected with pVSV-G (VSV glycoprotein expression plasmid), pRSV.REV (Rev expression plasmid), pMDLg/p.RRE (Gag/Pol expression plasmid), and pELNS transfer plasmid using Lipofectamine 2000 (Invitrogen). The viral supernatant was harvested at 24 and 48 h post-transfection. Viral particles were concentrated and resuspended in 0.5 ml by ultracentrifugation for 2.5 h at 25,000 rpm with a Beckman SW28 rotor (Beckman Coulter, Fullerton, CA).
4
Lentiviral Knockdown of TERT in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lentivirus vector was constructed using the LV-008 plasmid (Forevergen Biosciences Co., Ltd., Guangzhou, China) with a verified short hairpin RNA against TERT (shTERT) inserted, the pMD2.G plasmid (cat. no. 12259; Addgene, Inc., Cambridge, MA, USA) expressing the vesicular stomatitis virus glycoprotein gene and psPAX2 plasmid (cat. no. 12260; Addgene, Inc.) carrying the gag/pol-gene. For viral packaging, 8 µg shRNA plasmid, 2 µg pMD2.G plasmid and 4 µg psPAX2 plasmid were co-transfected using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) into 293T cells plated in 100-mm dishes. Transfected lentiviruses were collected 72 h following transfection and concentrated via ultra-centrifugation at 87,000 × g for 180 min at 4°C using a Beckman SW28 rotor (Beckman Coulter, Inc., Brea, CA, USA). SCC25 and UM1 cells were cultured with RPMI-1640 and seeded in a six-well plate. When cells reached 50–70% confluence, cells were infected with hTERT-lentivirus or a negative control (NC)-shRNA lentivirus, respectively, in the presence of 5 µg/ml polybrene (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). At 5 days following transfection, the knockdown efficiency was examined by reverse transcription (RT)-qPCR and western blotting. To select for stably silenced cells, puromycin (2 µg/ml; Sigma-Aldrich; Merck KGaA) was added to the medium.
5
Isolation and Purification of Xcc Bacteriophage Murka
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Xcc bacteriophage Murka was isolated from a soil sample taken from a field with a massive cabbage black rot outbreak, near Tiraspol (Transnistria, Moldova), in 2012. The phage was propagated using the Xcc Tr1 strain at 26 °C, in accordance with a previously published protocol [32 (link)]. Phage lysate was treated with chloroform, and bacterial debris were pelleted by centrifugation at 8000× g for 20 min, followed by filtration of the supernatants through 0.22 μm pore-size membrane filters (Millex-GV, Millipore, Cork, Ireland) and the addition of DNAse I (0.5 mg/mL, 1 h; Evrogen, Moscow, Russia). Phage filtrates were concentrated by ultracentrifugation at 100,000× g at 4 °C for 2 h, using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Phage purification was performed by ultracentrifugation in a CsCl step gradient (0.5–1.7 g/mL) at 22,000× g for 2 h. The phage-containing opalescent band was collected and dialysed against an SM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgSO4, 100 mM NaCl). The phage suspension was stored at 4 °C.
6
Isolation and Purification of Bacteriophage Ayka
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteriophage Ayka specific to C. flaccumfaciens pv. flaccumfaciens was isolated from a soil sample taken from the field in the Penza region. The phage was propagated using Cff strain C089 at 26 °C, according to a previously published protocol [35 ]. Phage lysate was treated with chloroform and bacterial debris was pelleted by centrifugation at 8000× g for 20 min, followed by filtration of the supernatants through 0.22 µm pore-size membrane filters, (Millex-GV, Millipore, Cork, Ireland) and the addition of DNAse I (0.5 mg/mL, 1 h; Evrogen, Moscow, Russia). Phage filtrates were concentrated by ultracentrifugation at 100,000× g at 4 °C for 2 h, using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Phage purification was performed by ultracentrifugation in CsCl step gradient (0.5–1.7 g/mL) at 22,000× g for 2 h. Phage-containing opalescent band was collected and dialysed against SM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgSO4, 100 mM NaCl). Phage suspension was stored at 4 °C.
7
Virus Purification from Allantoic Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Allantoic fluids were clarified by centrifugation at 4000 rpm using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific) at 4 °C for 30 min. Viruses in the allantoic fluid were pelleted through a 20% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4) by centrifugation in a Beckman L7–65 ultracentrifuge at 25,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Supernatants were aspirated and the pellets were re-suspended in PBS (pH 7.4). The protein content was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).
8
Polysome Profiling: Unveiling Translational Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polysome profiling was performed according to a published procedure [23 (link)]. Following centrifugation at 1200 RPM for 10 min, the cells were homogenized in lysis buffer [100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 µg/mL cycloheximide (Applichem, Darmstadt, Germany) and 30 U/mL RNase Inhibitor (Promega, Madison, WI, USA)]. The cell pellets were sheared to homogenization by passing the lysate through a 26 G needle at least 15 times and incubated on ice for 8 min. The lysates were then centrifuged at 12,000× g at 4 °C for 8 min. The supernatants were layered over 5–70% (w/v) sucrose gradients [100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl (pH 7.0), 200 U RNase Inhibitor (Promega, Madison, WI, USA)] prepared by using an ISCO Teledyne (Lincoln, NE, USA) density gradient fractionation system and centrifuged at 27,000 RPM for 2 h 55 min at 4 °C in a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Fractions were collected using the ISCO Teledyne density gradient fractionation system while reading absorbance at A254. Fractions were pooled as mRNP, monosomal, light and heavy polysomal sub-groups based on A254 readings. The total RNA was phenol-extracted from fractions by using phenol-chloroform-isoamyl alcohol (25:24:1) (Applichem, Darmstadt, Germany).
9
Virus Concentration and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viruses in the allantoic fluid were first inactivated using 0.05% beta-propiolactone (BPL) as described previously [23 ]. To concentrate the viruses, allantoic fluids were clarified by centrifugation at 4,000 rpm at 4°C for 30 min using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). Clarified allantoic fluids were laid on top of a 20% sucrose cushion in PBS (Gibco). Ultracentrifugation in a Beckman L7–65 ultracentrifuge at 25,000 rpm for 2 hours at 4°C using a Beckman SW28 rotor (Beckman Coulter, CA, USA) was performed to pellet the viruses through the sucrose cushion while soluble egg proteins were removed. The virus pellets were re-suspended in PBS (pH 7.4). The total protein content was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).
10
Virus Purification from Allantoic Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viruses in the allantoic fluid were first inactivated using 0.05% beta-propiolactone (BPL) as described previously (18 (link)). To concentrate the viruses, allantoic fluids were clarified by centrifugation at 4,000 rpm at 4°C for 30 min using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). Clarified allantoic fluids were laid on top of a 20% sucrose cushion in PBS (Gibco). Ultracentrifugation in a Beckman L7-65 ultracentrifuge at 25,000 rpm for 2 h at 4°C using a Beckman SW28 rotor (Beckman Coulter, CA, USA) was performed to pellet the viruses through the sucrose cushion while soluble egg proteins were removed. The virus pellets were re-suspended in PBS (pH 7.4). The total protein content was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!