Cell pellets were resuspended in 4 mL g−1 of cell pellet in buffer A (10 mM sodium phosphate, pH 7.5, 125 mM NaCl, 3 mM KCl) containing 10 mM benzamidine, 2 EDTA-free protease inhibitor cocktail tablets (Roche Diagnostic), 1 mM MgCl2, 20 μg/mL DNase, and 10 μg/mL lysozyme. Cells were stirred at 4 °C for 1 h and then passaged twice through a Thermo Spectronic French pressure cell at 18 000 psi. The cell debris was removed by centrifugation at 12 000 rpm for 15 min in a Beckman JA17 rotor. The resulting lysate was then centrifuged for 1 h at 25 000 rpm in a Beckman JA25.50 rotor to pellet the total cellular membranes. The pelleted membranes were combined and resuspended in 1 mL g−1 of Buffer A with an added EDTA-free protease inhibitor tablet, and the cytoplasmic membrane proteins were solubilized by addition of 2% (w/v) Triton X-100 (Sigma) and 0.5% (w/v) Sarkosyl (Teknova). The membrane fractions were stirred at room temperature for 1 h and repelleted at 25 000 rpm for 1 h in a Beckman JA25.50 rotor. The resulting supernatant containing only the cytoplasmic membrane proteins was discarded. The pelleted OM fraction was then resuspended in 30 mL of Buffer A containing a mini EDTA-free protease inhibitor cocktail tablet (Roche Diagnostic) and stirred at 4 °C overnight.
Ja25.50 rotor
Manufactured by Beckman Coulter
Sourced in United States
The JA25.50 rotor is a fixed-angle centrifuge rotor designed for use with Beckman Coulter centrifuges. It is capable of reaching a maximum speed of 50,000 revolutions per minute (rpm) and can generate a maximum relative centrifugal force (RCF) of 224,000 x g. The rotor is suitable for a variety of applications that require high-speed centrifugation, such as the separation of subcellular organelles, proteins, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
70ti rotor, by Beckman Coulter (2 mentions)
Ja 10 rotor, by Beckman Coulter (2 mentions)
Superdex 75 16 600 column, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Avanti j 25i, by Beckman Coulter (2 mentions)
Sw 32 ti swinging rotor, by Beckman Coulter (2 mentions)
Type 50.2 ti rotor, by Beckman Coulter (2 mentions)
Hiload 16 60 superdex 200 sec, by GE Healthcare (2 mentions)
Monoq 10 100 gl, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Tl 100 ultracentrifuge, by Beckman Coulter (1 mentions)
Protease inhibitor tablet, by Merck Group (1 mentions)
French pressure cell, by Thermo Fisher Scientific (1 mentions)
Ja 17 rotor, by Beckman Coulter (1 mentions)
Sarkosyl, by Teknova (1 mentions)
Mini edta free protease inhibitor cocktail tablet, by Roche (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail tablet, by Roche (1 mentions)
Type 70 ti rotor, by Beckman Coulter (1 mentions)
42 protocols using ja25.50 rotor
1
Outer Membrane Protein Extraction
2
Purification of Inclusion Bodies from Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested by centrifugation and pellets were resuspended in lysis buffer (Tris–HCl 50 mM pH 8; EDTA 10 mM) and sonicated for 15 min; the sonicator (MISONIX MOD.XL2020) was set up at 35% of amplitude with pulses of 30″ on and 30″ off. The lysate was centrifuged at 15,000 rpm for 60 min (JA25.50 rotor; Beckman). Inclusion bodies were resuspended in Tris–HCl 100 mM pH 8, EDTA 10 mM, Triton X-100 2%, urea 2 M, sonicated for 2 min and centrifugated for 30 min at 15,000 rpm (JA25.50 rotor; Beckman); this step was repeated twice. After 3 washings in Tris–HCl 100 mM pH 8 (preceded each time by 2 min sonication and centrifugation for 30 min), inclusion bodies were resuspended in denaturation buffer (Tris–HCl 50 mM pH 8, Gu-HCl 6 M) and sonicated for further 15 min. Inclusion bodies were then kept under shaking at 37 °C for 16 h after adding β-mercaptoethanol 20 mM.
3
Outer Membrane Vesicles Affect Amoeba Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OMVs were isolated by ultracentrifugation, as described previously [16 (link)]. V. cholerae strains were grown in broth culture to late exponential phase. Broth cultures were then centrifuged at 8,000 g (30 min, 4°C) in a JA-25.50 rotor (Beckman Instruments Inc.). Filtered (0.22 μm; Millipore) supernatants were centrifuged at 85,000 g (2 h, 4°C) in a 70 Ti rotor (Beckman Instruments Inc.) to collect OMVs. Pellets were washed twice with PBS, suspended in PBS to a total volume of 500 μL, and used as the OMVs preparation. Concentration of total protein in the OM vesicles was measured spectrophotometrically by the Bradford assay (Bio-Rad).
The effect of outer membrane vesicles on the viability of A. castellanii was examined by incubation of 50 μL amoeba cell suspension containing 106 cell/mL with 50 μL OM vesicle preparation from each bacterial strain or with 50 μL BPS for controls. Triplicate experiments were performed and the viability of amoebae was examined after 2 hours by viable count utilizing erythromycin B stain (ATCC).
The effect of outer membrane vesicles on the viability of A. castellanii was examined by incubation of 50 μL amoeba cell suspension containing 106 cell/mL with 50 μL OM vesicle preparation from each bacterial strain or with 50 μL BPS for controls. Triplicate experiments were performed and the viability of amoebae was examined after 2 hours by viable count utilizing erythromycin B stain (ATCC).
4
Preparation of Fibroblast Cell Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare extracts, primary fibroblast cells from two affected patients and an unaffected sibling were grown to confluence in two 10-cm diameter plates. Cells were washed twice with cold PBS and resuspended in 500 µl buffer A (10 mm HEPES–KOH pH 7.6, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm DTT, 0.5 mm PMSF, 2 mm Benzamidine, 1 µm Leupeptin, 2 µm Pepstatin A, 4 µm Chymostatin, 2.6 µm Aprotinin). Cell were broken in a Dounce homogenizer and lysates were centrifuged at 3000g (Beckman JA-25.50 rotor) for 5 min at 4°C, 450 µl of the supernatant were collected, mixed with 50 µl of 2 m KCl and centrifuged at 20 000g in a table-top Eppendorf centrifuge for 2 min at 4°C. The supernatant was further centrifuged at 110 000g for 20 min at 4°C in TLA 120.2 rotor in Beckman TL-100 Ultracentrifuge. Supernatants were transferred in dialysis tubing (MWCO 12–14 000 Daltons) and dialyzed two times, 60 min each; against 2 l of cold buffer D (20 mm HEPES–KOH, pH 7.6, 50 mm KCl, 0.2 mm EDTA, 0.5 mm DTT, 20% glycerol, 0.5 mm PMSF, 2 mm benzamidine). The resulting extracts were aliquoted and stored at −80°C. Protein concentrations of cleared lysates were measured using the Bradford assay.
5
Ribosome Isolation from Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown to exponential phase, pelleted by centrifugation, washed with water, and then snap-frozen in liquid nitrogen. Cells were incubated in lysis buffer (20 mM HEPES at pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate, 3 mM DTT, protease inhibitor tablets, 1 mg/mL zymolyase) for 5 min at 4°C and then lysed by French press (Sim-Aminco). The lysate was cleared by centrifuging at 19,000 rpm (Beckman Coulter, JA25.50 rotor) for 20 min at 4°C. The cleared lysates were applied to a 30% sucrose gradient containing 20 mM HEPES (pH 7.4), 500 mM potassium acetate, 2 mM magnesium acetate, and 3 mM DTT. Ribosomes were pelleted by centrifugation at 50,000 rpm (Beckman Coulter, type 70 Ti rotor) for 4 h at 4°C. The pellet was dissolved in lysis buffer and analyzed by SDS-PAGE.
6
Overnight Bacterial Culture Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Overnight (16 h) cultures were pre-grown at 37 °C in LB medium supplemented with 50 μg/mL kanamycin in the case of strains transformed with pSC101-based expression plasmids. Overnight cultures were diluted in filtered LB (33 mL cultures) and after 24 h growth at 18 °C harvested by pouring into precooled centrifuge bottles and pelleting at 10,000 rpm for 10 min at 4 °C (Beckman JA25.50 rotor). For the sake of convenience, cultures were diluted to different starting densities (Table S3) to ensure that all of them reach OD600 ≈ 0.5 simultaneously.
7
Ribosome Isolation from Bacterial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The overnight cultures were diluted 100-fold in LB media supplemented with 100 µg/ml of ampicillin and 30 µg/ml of spectinomycin. After reaching the A600 = ~0.5, cells were harvested by centrifugation at 5000 rpm for 10 min (4 °C) in the JA25.50 rotor (Beckman). Pellets were resuspended in ice-cold lysis buffer 2 (20 mM Tris–HCl, pH 7.5, 15 mM MgCl2, 1 mg/ml lysozyme). After 5 min incubation on ice, sodium deoxycholate (Sigma) was added to the final concentrations of 0.3% (w/w) and 2 units of RQ1 DNase (Promega) were added to the buffer. After 3 min incubation in ice, cell debris was harvested by centrifugation at 20,000g for 15 min (4 °C). Supernatant was loaded on the sucrose cushion (20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 500 mM NH4Cl, 0.5 mM EDTA, 2 mM β-mercaptoethanol, 30% (w/w) sucrose) and centrifuged for 90 min at 213,000g (4 °C) in the TLS-55 rotor (Beckman). Ribo-T or ribosome pellets were resuspended in water and rRNA was isolated by phenol–chloroform extraction and ethanol precipitation.
8
Purification of Halobacterium salinarum PM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Halobacterium salinarum S9 was grown as previously described, with no additional antibiotics necessary due to the culture conditions being too harsh for any contamination34 (link). The expression culture was grown at 38 °C with illumination in either an Erlenmeyer flask or a Winpact Bench-Top Fermenter (Major Science, Taiwan). The PM was purified from the bacterial pellet using a standard sucrose gradient procedure, in an Optima XE-90 Ultracentrifuge with the SW28 swing bucket rotor (Beckman Coulter, USA) spinning at 87300 × g for 22 h (Supplementary Figure S1 ). After the overnight centrifugation, the PM was extracted and underwent a fast centrifugation in an Avanti J-26S XP Centrifuge with JA25.50 rotor (Beckman Coulter, USA) at 27216 × g for 1 h to remove any residual sucrose. The PM pellet was then resuspended in water and gently sonicated using a Q700 sonicator (QSonica, USA). The concentration of PM was subsequently estimated with steady-state spectroscopy, using the absorption at 568 nm and the extinction coefficient of bR ε568 = 62700 M−1 cm−135 (link). The PM was then stored at 4 °C.
9
Membrane Preparation and Protein Purification
10
Purification of Pre-60S Ribosomal Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pelleted (1,850 g) for 10 min at 4 °C and washed once with PBS, cells were re-suspended in lysis buffer [50 mM HEPES (pH = 7.8), 60 mM KCl, 5 mM MgOAc, 0.05% NP-40, 1x cocktail Protease Inhibotor, 1 mM NaF, 5% glycerol and 40U RNasIN Ribonuclease Inhibitor (N2111, Promega)] and lysed by high pressure cell disruptor (JNBIO). The lysate was cleared for 30 min at 25,000 g in JA25.50 rotor (Beckman Coulter). Supernatants were collected and incubated with equilibrated ANTI-FLAG M2 Affinity Agarose Gel (Sigma-Aldrich) for 2 h, followed by five times washing with 20 c.v. (column volume) wash buffer [50 mM HEPES (pH = 7.8), 60 mM KCl, 5 mM MgOAc, 0.05% NP-40, 1x cocktail Protease inhibitor, 1 mM NaF] (100 c.v. totally) and eluted with 2 mg/ml 3XFLAG peptide in 5 c.v. wash buffer. Eluate was loaded onto a sucrose cushion buffer [40 mM HEPES (pH = 7.8), 90 mM KCl, 5 mM MgOAc and 30% w/v sucrose] in volume ratio of 10:9, and pre-60S particles were concentrated via centrifugation at 100,000 rpm in a TLA100 rotor (Beckman Coulter) for 23 mins and re-suspended in ribosome buffer [40 mM HEPES (pH = 7.8), 90 mM KCl, 5 mM MgOAc].
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!