Purification of OMPs from S. maltophilia was carried out as described by Hobb et al. [35] Briefly, cells from 250 mL culture of S. maltophilia ATCC13637 or M30 were resuspended in 7 mL of 10 mM HEPES, pH 7.4, and lysed by passing the culture twice through a French Press (Thermo Electron Corporation) at 1000 p.s.i. (6.9 MPa; 40K cell). The lysed cell preparation was centrifuged at 10000 x g for 10 min at 4C to remove cell debris. The membranes were then collected by ultracentrifugation at 100000 x g for 1 h at 4C (Beckman Ti70.1 rotor). The pellet was then resuspended in 2 mL 10 mM HEPES, pH 7.4, and washed in a total volume of 10 mL HEPES 10 mM, pH 7.4 and collected again by ultracentrifugation as described previously. The final pellet was resuspended in 5 mL 1% (w/v) N-laurylsarcosine (Sigma Aldrich) in 10 mM HEPES, pH 7.4, and incubated at 37C for 30 min with gentle shaking. The sarkosyl-treated membranes were then collected at 100000 x g for 1 h at 4C (Beckman, Ti70.1 rotor) and the resulting pellet was washed with 10 mL of HEPES 10 mM, pH 7.4. Following the final ultracentrifugation, the pellet was resuspended in 500 L of lysis solution (7 M urea, 2 M thiourea, 2.5% w/v CHAPS, 2% ASB-14 w/v, 0.5% pharmalytes, pH 3-10 and traces of bromophenol blue).
Ti70.1 rotor
Manufactured by Beckman Coulter
Sourced in United States
The Ti70.1 rotor is a high-speed ultracentrifuge rotor designed for use with Beckman Coulter ultracentrifuges. It has a maximum speed of 70,000 rpm and is capable of generating a maximum relative centrifugal force (RCF) of 500,000 x g. The rotor is constructed of titanium for increased durability and corrosion resistance.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Percoll reagent, by GE Healthcare (1 mentions)
Biosep sec s3000 column, by Phenomenex (1 mentions)
Econo pac, by Bio-Rad (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
Qubit protein assay, by Thermo Fisher Scientific (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
Beckman 70.1 ti rotor, by Beckman Coulter (1 mentions)
Sw32ti rotor, by Beckman Coulter (1 mentions)
Ti70 rotor, by Beckman Coulter (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Sf9 cells, by Thermo Fisher Scientific (1 mentions)
Triton x 114, by Merck Group (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Neb buffer 2, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Nalgene filter, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sodium lauryl sarcosine, by Merck Group (1 mentions)
Le 80, by Beckman Coulter (1 mentions)
21 protocols using ti70.1 rotor
1
Purification of Outer Membrane Proteins
2
In Vitro Recombinant Vault Synthesis
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In vitro translation was carried out using an insect-based cell-free system (EasyXpress Insect Kit II, Qiagen), using the protocol described by the manufacturer. In vitro synthesized recombinant vaults were further treated with RNase A and purified over 20% sucrose cushion (centrifuged at 100 000g for 2 h at 4 °C in Beckman Coulter Ti 70.1 rotor). Pellets were then dissolved in buffer A and visualized by EM.
3
EGFR Trafficking and Endosomal Profiling
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The cells were pretreated with 20 μM 6r for 1 h and subsequently incubated with 100 ng/mL EGF for 1 h further. After washing twice with phosphate-buffered saline (PBS), the cells were scraped off with 2 mL lysis buffer (10 mM ethanolamine, 1 mM EDTA, and 0.25 M sucrose, pH 7.2) and centrifuged at 200 × g for 10 min. The supernatant was then transferred into a new tube, and the pellet was resuspended with 2 mL homogenisation buffer. After additional centrifugation at 200 × g for 10 min, the supernatant was pooled, yielding 4 mL of postnuclear supernatant. Subsequently, the postnuclear supernatant was diluted with a 90% Percoll reagent (#17-0891-02; GE Healthcare) to a final concentration of 8 mL 10% or 25% Percoll. Percoll-containing samples were centrifuged for 25 min at 4 °C at 50 000 × g in a Beckman Ti70.1 rotor. Finally, the gradients were fractionated into 16 fractions from the top to the bottom. The fractions were then immunoblotted for an EGFR, an early endosomal marker (EEA1), a late endosomal marker (Rab7), and a lysosomal marker (LAMP2), as indicated in the figure.
4
Cellular Fractionation and Organelle Isolation
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Cells used for fractionation experiments were washed and resuspended in PBS (pH 7.4). Cells were disrupted by 20 passages through a 27-gauge needle followed by centrifugation for 2 min at 1,000 x g to pellet cellular debris and nuclei. The cell lysate was centrifuged for 10 min at 10,000 x g (4 °C) to pellet crude mitochondria (mitochondria and mitochondria-associated membranes (MAM)). Crude mitochondria were resuspended in a solution of 50 mM Tris-HCl (pH 7.4), 250 mM sucrose. The supernatant was centrifuged at 100,000 ×g for 60 min in a Beckman Ti-70.1 rotor at 4 °C to pellet microsomes, which were resuspended in 50 mM Tris-HCl (pH 7.4), 250 mM sucrose.
5
Syt1 KD PC12 Membrane-Based γ-Secretase Assay
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The crude homogenates of cellular membranes were prepared from parental or Syt1 KD PC12 cells in 20 mM HEPES (pH 7.4) containing protease inhibitor cocktail (Roche, Indianapolis, IN). The lysates were centrifuged at 3000xg for 15 minutes to remove cellular debris and nuclei. The supernatant was collected and centrifuged further at 100,000xg for 1 hour in a L8-80 M ultracentrifuge equipped with a Ti70.1 rotor (Beckman). Equal amounts of 1 % CHAPSO-solubilized membranes were incubated with C100-FLAG as a substrate (kindly provided by Dr. M.S. Wolfe, BWH, Boston, MA) for 4 hours at 37 °C. After the incubation, the samples were placed on ice to stop the reaction. For control purposes the reaction was carried out at 4 °C or in the presence of γ-secretase inhibitors.
6
Purification of Membrane-bound CybB Proteins
7
Membrane Protein Extraction Protocol
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Cell pellets were thawed on
ice, resuspended in cold lysis buffer [50 mM Tris-HCl pH 8, 2 mM dithiothreitol
(DTT), 1 mM MgCl2, 2.5 U/mL benzonase], and incubated on
ice for 30 min. The cytosolic fraction (supernatant) was separated
from the membranes by ultra-centrifugation (100,000g, 45 min, 4 °C, Beckman Coulter, Ti 70.1 rotor). The pellet
(membrane fraction) was resuspended in cold storage buffer (50 mM
Tris-HCl pH 8, 2 mM DTT) and homogenized by thorough pipetting and
passage through an insulin needle (29G). Protein concentrations were
determined by a Quick Start Bradford protein assay (Bio-Rad) or Qubit
protein assay (Invitrogen). The samples were flash-frozen in liquid
N2 and stored at −80 °C.
ice, resuspended in cold lysis buffer [50 mM Tris-HCl pH 8, 2 mM dithiothreitol
(DTT), 1 mM MgCl2, 2.5 U/mL benzonase], and incubated on
ice for 30 min. The cytosolic fraction (supernatant) was separated
from the membranes by ultra-centrifugation (100,000g, 45 min, 4 °C, Beckman Coulter, Ti 70.1 rotor). The pellet
(membrane fraction) was resuspended in cold storage buffer (50 mM
Tris-HCl pH 8, 2 mM DTT) and homogenized by thorough pipetting and
passage through an insulin needle (29G). Protein concentrations were
determined by a Quick Start Bradford protein assay (Bio-Rad) or Qubit
protein assay (Invitrogen). The samples were flash-frozen in liquid
N2 and stored at −80 °C.
8
Ribosome Isolation and Purification
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Ribosomes were isolated essentially as described59 . In brief, The Δrnr strain (BGSC BKE33610 trpC2; Δrnr::erm) was grown in 2 l LB medium until OD600 nm = 0.8. The cells were flash-frozen in ribosome buffer (20 mM HEPES-KOH, pH 7.5, 6 mM magnesium acetate, 30 mM NH4Cl and 0.5 mM TCEP) and lysed under cryogenic conditions using a Retsch MM400 (Retsch). The lysate was pre-cleared at 17,000 rpm for 30 min at 4 °C. The supernatant was then centrifuged for 17 h at 40,000 rpm in a Beckman 70.1 Ti rotor to pellet 70S ribosomes and remaining polysomes. The crude ribosomes were resuspended by gentle shaking at 4 °C for 60 min in either ribosome or dissociation buffer (20 mM HEPES-KOH, pH 7.5, 1 mM magnesium acetate, 200 mM NH4Cl and 0.5 mM TCEP) for subsequent isolation of 70S or ribosomal subunits, respectively. The resuspended ribosomes were subsequently loaded on 10–30% sucrose gradients and run in a Beckman SW32Ti rotor at 18,000 rpm for 19.5 h. The peaks corresponding to 30S, 50S and 70S were collected and the 70S ribosomes, or subunits, were pelleted further at 40,000 rpm for 22 h in a Beckman Ti70 rotor, or at 47,000 rpm for 20 h in a Beckman Ti70.1 rotor.
9
Purification of LH2 and RC-LH1 Proteins
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Wild type LH2 and ΔcrtB RC-LH1 proteins were purified as described previously [55 (link),56 (link)]. Briefly, cells were grown in 1.5 L M22+ medium [57 (link)] under semi aerobic conditions (in 2 L conical flasks shaken at 180 RPM at 34°C in darkness) for 72 h. Cells were harvested at 4,000 × g and broken via two passes through a French pressure cell (AmInCo, USA) at 18,000 psi, then unbroken cells and insoluble debris were removed by centrifugation at 25,000 × g for 15 min at 4°C. The supernatant was loaded onto a 40/15% w/w sucrose gradient and centrifuged at 100,000 × g for 10 h at 4°C in order to isolate the intracytoplasmic membranes (ICM). After harvesting, the ICMs were solubilised by addition of 3% (w/v) β-DDM for RC-LH1, or in 4% N,N-dimethyldodecylamine-N-oxide (LDAO) for LH2, stirring in the dark at 4°C for 45 min. The solubilized membrane solution was diluted at least three-fold in working buffer and centrifuged for 1 hour in a Beckman Ti 70.1 rotor at 48,000 rpm (160,000 × g ) at 4°C to remove unsolubilized material. The supernatant was further purified by using ion-exchange chromatography and concentrated using Amicon 100,000 MWCO spin filters (Millipore) in 10 mM HEPES pH 7.8, 50 mM NaCl, 0.03% (w/v) β-DDM buffer.
10
Isolation of LDL/VLDL and HDL by Ultracentrifugation
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LDL/VLDL and HDL were isolated from EDTA plasma by density-gradient ultracentrifugation as previously described [27 (link)]. In short, plasma was mixed with a sucrose and EDTA solution and overlaid with a KBr/PBS solution with density 1.063 g/ml. Centrifugation was performed at 290000×g at 15°C for 4 h in a Beckman Coulter Ti 70.1 rotor (Beckman Instruments, Inc., Palo Alto, CA). The LDL/VLDL and HDL fractions were extracted separately and mixed with KBr/PBS (density 1.24 g/ml) before being subjected to a second round of ultracentrifugation with the same conditions for 2 h. LDL/VLDL and HDL were aspirated from the top of the tubes and desalted using PD-10 desalting columns (GE Healthcare, Little Chalfont, U.K.).
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