Jla 8.1000 rotor

Manufactured by Beckman Coulter

Sourced in France

The JLA 8.1000 rotor is a centrifugation accessory designed for Beckman Coulter laboratory equipment. It is a fixed-angle rotor capable of high-speed centrifugation. The rotor can accommodate up to 8 sample tubes or bottles with a maximum volume of 1000 milliliters each.

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Lab products found in correlation

70ti rotor, by Beckman Coulter (2 mentions) Bl21 de3 plyss competent cells, by Promega (2 mentions) Tryptic soy broth medium, by Merck Group (1 mentions) Sheep blood, by bioMérieux (1 mentions) Columbia agar medium, by bioMérieux (1 mentions) Avanti j 26 xp centrifuge, by Beckman Coulter (1 mentions) Jla 10.500 rotor, by Beckman Coulter (1 mentions) Pgex 6p 2 vector, by GE Healthcare (1 mentions) Avanti j 26 xpi centrifuge, by Beckman Coulter (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions) L2630, by Merck Group (1 mentions) Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions) L9143, by Merck Group (1 mentions) Xk 16 20 column, by GE Healthcare (1 mentions) Kta start system, by GE Healthcare (1 mentions) Talon affinity resin, by Generon (1 mentions) Ja25.50 rotor, by Beckman Coulter (1 mentions) Columbia agar 5 sheep blood, by bioMérieux (1 mentions)

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JLA 8.1000 (11 citations)

13 protocols using jla 8.1000 rotor

Cultivation and Extraction of Bacterial Strains

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The strains were stored at −80 °C. The cultures were grown on solid Columbia agar medium with 5% sheep blood (bioMérieux) under aerobic conditions at 32 °C. Thereafter, several colonies were transferred in sterile conditions into 1 L of liquid Tryptic soy broth medium (Sigma-Aldrich, France), which was incubated for 3 days for Bacillus spp. and for 14 days for Streptomyces spp. at 32 °C and 110 rpm in a shaker incubator under aerobic conditions.
The supernatant-pellet separation was carried out by centrifugation at 8000× g for 20 min at 4 °C using an A98813 J-Lite PP bottle assembly with a JLA-8.1000 rotor (Beckman Coulter, Villepinte, France). After centrifugation, the supernatant was immediately filtered through a 0.45 μm filter and placed into 75 mL flasks, after which it was frozen horizontally overnight at −80°C and then lyophilized the next day. The lyophilizate was stored at −20°C prior to the assays.

Dahmana H., Sambou M., Raoult D., Fenollar F, & Mediannikov O. (2020). Biological Control of Aedes albopictus: Obtained from the New Bacterial Candidates with Insecticidal Activity. Insects, 11(7), 403.

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Optimized Expression of TetA(B) Constructs

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Expression of TetA(B)-His10 and TetA(B)-Δ394–401-Thrombin-His12 constructs were optimized in E. coli strain C43 [45,46] . Fresh transformations of each construct were made and plated on to 90 mm 2 × TY 1.5% (w/v) agar plates containing 100 μg/ml ampicillin. Colonies were picked into 10 ml of 2 × TY containing 100 μg/ml ampicillin and grown for 18 h in sterile test tubes at 37 °C and 250 rpm. 4 ml of this bacterial culture was used to inoculate 1 l of 2 × TY containing 100 μg/ml ampicillin in a 2 l conical flask, which was incubated at 37 °C, shaking at 220 rpm in an Infors HT Multitron standard shaker until the cell density reached an OD₆₀₀ of 0.3–0.5. IPTG was added to a final concentration of 0.2 mM and the temperature was lowered to 27 °C for 4 h. Cells were harvested in 1 l centrifuge bottles (Beckman Coulter) at 3063 ×g for 15 min at 4 °C in a JLA-8.1000 rotor (Beckman Coulter) in Avanti J-26 XP centrifuge (Beckman Coulter) and resuspended in 20 ml per litre of cells of 20 mM Tris pH 7.4 with protease inhibitor tablets made up to manufacturer's specifications, which were then frozen at − 20 °C in 50 ml Falcon tubes.

Wright D.J, & Tate C.G. (2015). Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B). Biochimica et Biophysica Acta, 1848(10), 2261-2270.

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Engineered CybB Expression and Purification

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The expression plasmid pCybB-His was engineered from a GFP-tagged E. coli CybB construct by replacing the GFP moiety with a 8x His-tag, as described previously38 . The new construct was transformed into chemically competent T7 express lysY (New England Biolabs) and grown at 37° C, either in LB supplemented with 25 mg/L kanamycin and 0.01% Antifoam 204 (for non-Se-Met labeled CybB) or in minimal media supplemented with 0.4% glucose, 2 mM MgSO₄, 0.1 mM CaCl₂, 1 μM MnCl₂, 10 μM FeSO₄, 25 mg/L kanamycin and 0.01% Antifoam 204 (for production of selenomethionine labeled protein for structural determination). At culture OD₆₀₀ ~0.7, the minimal media cultures were supplemented with a mixture of amino acids (100 mg/L lysine, 100 mg/L threonine, 100 mg/L phenylalanine 50 mg/L leucine, 50 mg/L isoleucine, 50 mg/L valine and 50 mg/L selenomethionine). Expression of CybB and CybB_SeMet was induced by the addition of 0.2 mM IPTG, and the culture was grown for 4 more hours. The cells were harvested by centrifugation at 7500 g in a JLA 8.1000 rotor (Beckman Coulter), transferred to 50 ml tubes and frozen at -20°C.

Lundgren C.A., Sjöstrand D., Biner O., Bennett M., Rudling A., Johansson A.L., Brzezinski P., Carlsson J., von Ballmoos C, & Högbom M. (2018). Scavenging of superoxide by a membrane bound superoxide oxidase. Nature chemical biology, 14(8), 788-793.

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Cryogenic Cell Grinding and Preservation

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Cell cultures in late exponential growth were collected by centrifugation at 5000 rpm for 15 min at 4°C in a JLA-8.1000 rotor (Beckman Coulter, High Wycombe, UK). Cell pellets were resuspended in one-quarter culture volume of wash buffer (10 mM NaPO₄ pH 7.5 and 0.5 mM EDTA) and then washed three times by centrifugation at 5000 rpm for 15 min at 4°C in a JLA-10.500 rotor (Beckman Coulter) and resuspension in the same volume of wash buffer. After the final centrifugation, the cell pellet was weighed and resuspended in wash buffer, using a ratio of 0.3 mL wash buffer per gram of cell pellet. The cell suspension was then quick-frozen by drop-wise addition into liquid nitrogen and stored at −80°C until further use.
Cryogrinding was performed using an RM100 electric mortar grinder with a zirconium oxide mortar and pestle (Retsch, Hope Valley, UK). The mortar and pestle were pre-cooled by filling with liquid nitrogen for 10 min before grinding. Frozen cells were then added into the pre-cooled grinder and ground for 40 min, with regular generous addition of liquid nitrogen to maintain the temperature and prevent cell clumping during the grinding process. Cryogrindate cell powder was recovered and stored at −80°C until further use.

Bao X.X., Spanos C., Kojidani T., Lynch E.M., Rappsilber J., Hiraoka Y., Haraguchi T, & Sawin K.E. (2018). Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore. eLife, 7, e33465.

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Overexpression and Purification of Listeria RsbR and RsbS

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The L. innocua genes CAC96120.1 (LiRsbR) and CAC96121.1 (LiRsbS) were cloned into the pET11a expression vector (Novagen, 69436-3) via NdeI and BamHI restriction sites. A ribosome binding site (construct from GenScript) was inserted between the coding sequences. The construct was transformed into E. coli DH5α for plasmid isolation and maintenance.
For over-expression, the pET11a plasmid was transformed into E. coli BL21 Star (DE3) cells. Expression cultures were grown to a starting OD₆₀₀ of 0.6–0.8, induced with IPTG (final concentration 1 mM) and harvested by centrifugation after 3 h of expression at 37 °C and 120 rpm. The cells were harvested by centrifugation in a JLA-8.1000 rotor (Beckmann Coulter) at 4000 rpm for 30 min, at 4 °C and stored at −80 °C. For LiRsbT a pGEX6P-2 vector (GE Healthcare) containing a GST-tag fused rsbT gene from L. innocua (CAC96122.1) was transformed into E. coli BL21 (DE3) cells and grown in LB media at 37 °C, 120 rpm until OD₆₀₀ = 0.6, then induced with 0.1 mM IPTG and allowed to express for 3 h. The cells were resuspended in Elution buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl) and lysis and clarification carried out as above.

Miksys A., Fu L., Madej M.G., Guerreiro D.N., Kaltwasser S., Conway M., Ejder S., Bruckmann A., Marles-Wright J., Lewis R.J., O’Byrne C., Pané-Farré J, & Ziegler C. (2022). Molecular insights into intra-complex signal transmission during stressosome activation. Communications Biology, 5, 621.

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Purification of His-tagged Yeast NAP1

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The gene for yeast NAP1 bearing a His-tag in pET28 was introduced in BL21(DE3)pLysS competent cells (Promega, cat. no. L1195). A 2 l culture was induced with 2 ml of 1 M IPTG for 5 h, harvested by centrifugation in the Beckman JLA 8.1000 rotor (see above) at 6000 rpm for 15 min, resuspended in 50 ml of Talon buffer (50 mM potassium phosphate pH 7.6, 100 mM KCl, 2 mM MgCl₂, 2 mM β-mercaptoethanol, 0.1% NP40, 0.5x protease inhibitors, 10% glycerol), frozen, thawed, supplemented with another 0.5x protease inhibitors, solubilized by passage through a French pressure cell, and centrifuged in a Beckman 70 Ti rotor at 50,000 rpm for 30 min. The supernatant was allowed to bind to 5 ml of Talon beads (equilibrated in Talon buffer) for 1 h at 4 °C, washed in a column with te n column volumes of 40 mM imidazole in Talon buffer, and eluted with three column volumes of 250 mM imidazole in Talon buffer.

Lorch Y., Maier-Davis B, & Kornberg R.D. (2018). Histone Acetylation Inhibits RSC and Stabilizes the +1 Nucleosome. Molecular cell, 72(3), 594-600.e2.

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His-tagged Nap1 Protein Purification

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The gene for yeast NAP1 bearing a His tag in pET28 was introduced in BL21(DE3)pLysS competent cells (Promega, cat. no. L1195). A 2 l culture was induced with 2 ml of 1 M IPTG for 5 h, harvested by centrifugation in the Beckman JLA 8.1000 rotor (see above) at 6000 rpm for 15 min, resuspended in 50 ml of Talon buffer [50 mM potassium phosphate (pH 7.6), 100 mM KCl, 2 mM MgCl₂, 2 mM β-mercaptoethanol, 0.1% NP-40, 0.5× protease inhibitors, 10% glycerol], frozen, thawed, supplemented with another 0.5× protease inhibitors, solubilized by passage through a French pressure cell and centrifuged in a Beckman 70 Ti rotor at 50 000 rpm for 30 min. The supernatant was allowed to bind to 5 ml of Talon beads (equilibrated in Talon buffer) for 1 h at 4°C, washed in a column with 10 column volumes of 40 mM imidazole in Talon buffer and eluted with 3 column volumes of 250 mM imidazole in Talon buffer.

Lorch Y., Kornberg R.D, & Maier-Davis B. (2023). Role of the histone tails in histone octamer transfer. Nucleic Acids Research, 51(8), 3671-3678.

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Recombinant Expression of SecA1 and SecA2

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E. coli BL21(λDE3) was transformed with the plasmids and used for expression and co-expression of SecA1 and SecA2. Cells were cultured at 30°C in LB supplemented with 34 μg/ml chloramphenicol for SecA1 expression or with 100 μg/ml ampicillin for SecA2 expression. For co-expression of SecA1 and SecA2, cells were cultured at 30°C in LB supplemented with 17 μg/ml chloramphenicol and 50 μg/ml ampicillin. Cells were grown to an optical density at 600 nm (OD₆₀₀) of 0.6. Protein expression was induced by adding 1 mM isopropyl-D-thiogalactopyranoside (IPTG), and cells were grown further for 2 hours. Cells were harvested using the Beckman JLA 8.1000 rotor (7000 rpm, 15 min, 4°C), resuspended in 25 mM HEPES-KOH pH 6.5 and stored at -80°C. E. coli SecA was produced as described [45 (link)].

Prabudiansyah I., Kusters I, & Driessen A.J. (2015). In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis. PLoS ONE, 10(6), e0128788.

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Recombinant Protein Expression in E. coli

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BL21(DE3) Escherichia coli cells were transformed with desired plasmids and cultured in LB media at 37 °C. At OD₆₀₀ 0.6 to 0.8, the culture temperature was lowered to 20 °C, IPTG (GoldBio) was added at 0.5 mM to induce protein expression and cultures further incubated at 20 °C overnight. The cells were harvested by centrifugation at 4000g for 20 min in an Avanti J-26 XPI centrifuge with a JLA 8.1000 rotor (Beckman Coulter).

Tao J., Berthet A., Citron Y.R., Tsiolaki P.L., Stanley R., Gestwicki J.E., Agard D.A, & McConlogue L. (2021). Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism. The Journal of Biological Chemistry, 296, 100613.

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Extraction and Purification of Bacterial LPS

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Commercial LPS from E. coli and P. aeruginosa (L2630 and L9143, respectively, Sigma-Aldrich, Madrid, Spain) were dissolved in Milli-Q water at 1 µg ml -1 and, subsequently, kept at -20ºC. In order to obtain A. hydrophila LPS, bacteria were grown on LB agar plates containing 50 µg ml -1 ampicillin. A single isolated colony was inoculated first in 50 ml LB overnight (O/N) culture to later grown O/N in 2.5 l of LB for the large-scale production. The A. hydrophila culture was centrifuged at 6,000 rpm, J o u r n a l P r e -p r o o f 4°C, 30 min in a Beckman JLA 8.1000 rotor. Then resuspended sequencially in PBS, ethanol, acetone and diethyl ether to finally prepare the dried biomass. Then a phenolwater extraction method was used to separate the LPS from the biomass into two phases after performing four centrifugations at 4ºC. The LPS was further dialyzed at 4ºC for seven days and its concentration was checked using NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) . The solution was diluted to obtain a final working 1 μg μl concentration.

Moraleda-Prados J., Caballero-Huertas M., Valdivieso A., Joly S., Ji J., Roher N, & Ribas L. (2021). Epigenetic differences in the innate response after immune stimulation during zebrafish sex differentiation. Developmental and comparative immunology, 114.

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