Avanti j 25i

Manufactured by Beckman Coulter

Sourced in United States

The Avanti J-25I is a high-performance centrifuge from Beckman Coulter. It is designed for efficient separation of biological samples, capable of reaching speeds up to 25,000 RPM and generating up to 50,000 x g of force. The Avanti J-25I features a temperature control system that can maintain sample temperatures between -20°C and 40°C.

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Lab products found in correlation

Ja25.50 rotor, by Beckman Coulter (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Odyssey scanner, by LI COR (1 mentions) Irdye 680cw, by LI COR (1 mentions) Irdye 800cw, by LI COR (1 mentions) Le 80k, by Beckman Coulter (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) Optima le 80k ultracentrifuge, by Beckman Coulter (1 mentions) Histrap ff crude column, by GE Healthcare (1 mentions) Protease inhibitor cocktail set 3, by Merck Group (1 mentions) Ripa buffer, by Merck Group (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Dneasy powerlyzer microbial kit, by Qiagen (1 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Tandem 1081 pump, by Sartorius (1 mentions) Benzonase, by Merck Group (1 mentions) Bca assay kit, by Beyotime (1 mentions) Phenol, by Thermo Fisher Scientific (1 mentions)

15 protocols using avanti j 25i

Bacterial Expression of Glutathione-S-Transferase Fusion Proteins

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C41 and C43 bacteria used for protein production were obtained from Lucigen. C41 bacteria were used for glutathione-S-transferase (GST)-5-LO expression. C43 bacteria were transfected with expression vectors GST-ORMDL3, GST-LTC₄ synthase (LTC4S), GST-FLAP, or empty pGEX-4T-2 vector to express GST alone. All bacteria strains were cultured in Luria-Bertani broth in the presence of ampicillin (100 μg/ml) at 37°C until the absorbance at 600 nm reached 0.8–1.0. Cultures were then transferred to 12°C. Protein expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (final concentration of 1 mM) for 6 h. Bacteria were collected by centrifugation in Avanti J25I (Beckman Coulter) using JLA-10, 500 rotor at 8,000 rpm for 15 min at 4°C. Pellets were resuspended in an equilibration/washing (E/W) buffer containing 125 mM Tris-HCl, pH 8.0, 150 mM NaCl, and supplemented with 1% CHAPS, 1% Nonidet P-40, 1 mM PMSF, and a protein inhibitory cocktail from Promega. Bacteria were disrupted by 40 sonication cycles, each lasting 15 s, at amplitude 7, followed by 15 s pause on ice. Sonicates were centrifuged in a table centrifuge at 4°C, 13,200 g to remove the debris. Lysates were aliquoted and frozen in liquid nitrogen until further use.

Bugajev V., Paulenda T., Utekal P., Mrkacek M., Halova I., Kuchar L., Kuda O., Vavrova P., Schuster B., Fuentes-Liso S., Potuckova L., Smrz D., Cernohouzova S., Draberova L., Bambouskova M, & Draber P. (2021). Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways. Journal of Lipid Research, 62, 100121.

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Western Blot Analysis of Ciliary Proteins

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Half of the supernatant material from deciliated, serum-starved or -stimulated cells was centrifuged at 21,000 x g for 15 minutes in JA25.5 rotor in Beckman Coulter Avanti J-25I centrifuge at 4°C. The supernatant was carefully removed, and pellets were resuspended in 160 μl of sample buffer (1% SDS, 10 mM Tris-HCl, pH 7.5, 2 mM EDTA). Samples were boiled at 95°C for 8 mins, and equal volumes were separated by 10% PAGE and transferred to PVDF. Blots were blocked (2% BSA, 1% normal donkey and goat serum in TBS, pH 7.4) for 1 hr at RT or overnight at 4°C. Membranes were blotted with YL1/2 (1:1,000), mouse acetylated-tubulin antibody (1:1,000), and IFT88 rabbit antibody (1:500) in blocking buffer for 1 hr at RT. Blots were washed 5x with TBST. Secondary anti-rabbit, anti-mouse, or anti-rat antibodies labeled with either IRDye680CW or IRDye800CW (Li-Cor Biosciences, #926–32213), at 1:30,000 dilution were incubated with blots for 30 minutes at RT. Blots were washed five times with TBST and scanned on Licor Odyssey scanner (Li-Cor BioSciences).

Mirvis M., Siemers K.A., Nelson W.J, & Stearns T.P. (2019). Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding. PLoS Biology, 17(7), e3000381.

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TCUEAP1 Phage Purification Protocol

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Lysates for TCUEAP1 phage purification were prepared by infecting 200 ml of E. anophelis ANO15 in early log-phase with TCUEAP1 at a multiplicity of infection (MOI) of about 1.0, and incubating with aeration for 4 h. Crude lysates were centrifuged and the supernatants were passed through a membrane filter with a pore size of 0.45 μm. The phage particles were concentrated by centrifugation for 2 h at 39,000 × g in a Beckman Avanti J-25I. The pellets were re-suspended in 1.0 ml of TE buffer and purified by banding on the block gradient of CsCl representing 1.2, 1.3, and 1.4 g/cm³ (2 ml for each block) in ultracentrifugation. The ultracentrifugation conditions were 107,200 × g for 3 h at 4°C with the SW41Ti rotor in a Beckman LE-80K. The phage band collected was dialyzed against the TE buffer and then stored at 4°C until further use.

Peng S.Y., Chen L.K., Wu W.J., Paramita P., Yang P.W., Li Y.Z., Lai M.J, & Chang K.C. (2020). Isolation and Characterization of a New Phage Infecting Elizabethkingia anophelis and Evaluation of Its Therapeutic Efficacy in vitro and in vivo. Frontiers in Microbiology, 11, 728.

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Microalgal Lipid Extraction and Quantification

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F. diplosiphon B481-SD cultures were grown in BG11/HEPES medium amended with 3.2, 12.8 and 51.2 mg/L nZVIs and ascorbic acid at 2, 4, 6, 8, and 10 mM. Cultures were grown under continuous shaking at 170 rpm and 28 °C for a period of 15 days. Cells were centrifuged using a Avanti-J25I with a JA25.50 rotor (Beckman-Coulter, Pasadena, CA, USA), lyophilized overnight, and sonicated in 5 mL chloroform:methanol (2:1) for 30 s. Total lipids were extracted using a 2:1 chloroform: methanol mixture according to the method of Folch et al. [14 (link)]. The mixture was agitated for 15–20 min in an orbital shaker after dispersion at room temperature and the homogenate centrifuged to recover the liquid phase. The solvent was washed with 0.2 volumes (1 for 5 mL) distilled H2O, vortexed briefly, and centrifuged at 2000 rpm to separate the phases. The lower phase was transferred to a pre-weighed vial and the interface was rinsed twice using methanol:water (1:1) without mixing the whole preparation. The lower chloroform phase containing lipids was evaporated under vacuum in a rotary evaporator after centrifugation and siphoning, and the extracted lipids were used for transesterification [15 (link)]. Three replicates were maintained and the experiment repeated once.

Wyatt L., Gichuki S., Yalcin Y.S, & Sitther V. (2023). Impact of Ascorbic Acid on Zero-Valent Iron Nanoparticle and UV-B Mediated Stress in the Cyanobacterium, Fremyella diplosiphon. Microorganisms, 11(5), 1245.

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Bile Sample Collection and Processing

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Bile samples from both groups were collected at the time of percutaneous transhepatic biliary drainage (PTBD) for relieving cholangitis or obstructive jaundice (for CCA) and for relieving cholangitis by removing common bile duct stones (for the control). In both patients, bile fluid was collected after resolving cholangitis at least 3 days after the PTBD procedure. Collected bile fluid was centrifuged immediately at 16,000 ×g for 10 minutes at 4℃ to obtain supernatant (bile); each pellet was resuspended in chilled phosphate-buffered saline (2-fold) and then centrifuged at 16,000 ×g for 5 minutes at 4℃ to obtain a bile pellet, as described in the protocol by Abi Zabron Imperial College, London, England. All experiments were performed with Avanti J-25I (Beckman, Pasadena, CA, USA), and bile and pellets were stored at −80℃. After separation, we used these samples in all experiments.

Han J.Y., Ahn K.S., Kim Y.H., Kim T.S., Baek W.K., Suh S.I, & Kang K.J. (2021). Circulating microRNAs as biomarkers in bile-derived exosomes of cholangiocarcinoma. Annals of Surgical Treatment and Research, 101(3), 140-150.

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Purification of Phage Particles

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Phage lysates (200 ml, ca. 1.0 × 10¹⁰ PFU/ml) were centrifuged at 7,800 × g for 10 min. The supernatants were passed through a 0.45-μm-pore-size membrane filter and centrifuged at 22,000 rpm (BECKMAN COULTER Avanti-J25I) for 2 h at 4°C. The pellets were suspended in 1.0 ml of SM buffer (0.05 M Tris-HCl, pH 7.5 containing 0.1 M NaCl, 0.008 M MgSO₄‧7H₂O, and 0.01% gelatin) and loaded on block gradient of CsCl (ρ = 1.50, 1.48, 1.45, 1.43, and 1.40 g/cm³), followed by ultracentrifugation at 30,000 rpm for 3 h at 4°C with the SW41Ti rotor in a BECKMAN Optima LE-80K Ultracentrifuge. The banded phage particles were recovered, desalted with Amicon Ultra Centrifugal Filters (10,000 MWCO, Millipore Corporation, Ireland), and then stored at 4°C until used.

Wang J.B., Lin N.T., Tseng Y.H, & Weng S.F. (2016). Genomic Characterization of the Novel Aeromonas hydrophila Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family. PLoS ONE, 11(9), e0162060.

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Extraction and Purification of Myofibrillar Protein

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The extraction of MP was accorded to a previous study [18 (link)]. Beef semimembranosus (40 g) was homogenized at 12,000 rpm for 2 × 30 s with 200 mL of extraction buffer I (1 mM EGTA, 2 mM MgCl₂, 100 mM NaCl, and 10 mM potassium phosphate, pH 7.0). The buffer I was first pre-cooled in a refrigerator at 4 °C for 12 h, and buffer I was under ice–water during the homogenate process. Then, the homogenate was centrifuged (Avanti J-25i, Beckman Coulter, CA, USA) at 2000× g (4 °C, 15 min). The above operations were repeated three times using buffer I. The pellet was further homogenized (12,000 rpm, 2 × 30 s) with 200 mL of extraction buffer II (0.1 M NaCl, pH 6.0) under ice–water. The homogenate was filtered through two layers of gauze followed by centrifugation (4 °C, 2000× g, 15 min). The pellet was washed three times with buffer II to obtain MP. The obtained MP was solubilized in buffer III (0.6 M NaCl, 20 mM Na₂HPO₄/NaH₂PO₄, pH 6.0), and treated with MDA immediately. The MP concentration was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).

Yin Y., Zhou L., Cai J., Feng F., Xing L, & Zhang W. (2022). Effect of Malondialdehyde on the Digestibility of Beef Myofibrillar Protein: Potential Mechanisms from Structure to Modification Site. Foods, 11(15), 2176.

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Purification of His-tagged Recombinant Protein

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Sf9 pellets were resuspended in lysis buffer [20 mM imidazole pH 8.0, 100 mM NaCl, 5% glycerol, 2 mM βMe, protease (Protease Inhibitor Cocktail Set III, Sigma)] and lysed by sonication. Triton X-100 was added to 0.1% final, and lysate was centrifuged for 45 min at 20,000×g at 1 °C. (Beckman Coulter Avanti J-25I, JA 25.50 rotor). Supernatant was loaded onto a HisTrap FF Crude column (GE Healthcare) and superlooped for 1 h. The column was washed with Ni-NTA A buffer [20 mM imidazole pH 8.0, 100 mM NaCl, 5% glycerol (v/v), 2 mM βMe], washed with 6% Ni-NTA B buffer [30 mM imidazole pH 8.0, 100 mM NaCl, 5% (v/v) glycerol, 2 mM βMe], and the protein eluted with 100% Ni-NTA B buffer (450 mM imidazole). Elution fractions were passed through a 5 ml StrepTrapHP column pre-equilibrated in GF buffer [20 mM imidazole pH 7.0, 150 mM NaCl, 5% glycerol (v/v), 0.5 mM TCEP]. The column was washed with GF buffer before loading a tobacco etch virus protease containing a stabilizing lipoyl domain (Lip-TEV), and cleavage proceeded overnight. Cleaved protein was eluted with GF buffer and concentrated down to 250 µl in an Amicon 50 kDa MWCO concentrator (MilliporeSigma) pre-equilibrated in GF buffer. Concentrated protein was flash frozen in liquid nitrogen and stored at −80 °C.

Ulengin-Talkish I., Parson M.A., Jenkins M.L., Roy J., Shih A.Z., St-Denis N., Gulyas G., Balla T., Gingras A.C., Várnai P., Conibear E., Burke J.E, & Cyert M.S. (2021). Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane. Nature Communications, 12, 6064.

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Quantifying Cellulose Nanofibrils Yield

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Process yield was calculated as the washed gravimetric yield after the chemical treatment relative dry wood mass. The fraction of the process yield that comprised individual, colloidally stable nanofibrils were further quantified through centrifugation of the suspensions at 12,000× g (Avanti J25i, Beckman Coulter Inc. Brea, CA, USA) for 20 min at an approximate consistency of 0.2 wt %. The suspensions were decanted and the solids retained in the sediment were dried for 24 h at 95 °C. This was repeated three times and the nanofibril-fraction was then calculated according to the Equation (2), where m_p and m_s is the dry precipitate and supernatant mass in the sample, respectively. The nanofibril-fraction (

Φ

) was presented as fraction of the process yield.

Φ = 1 - \frac{m_{p}}{m_{p} + m_{s}}

Jonasson S., Bünder A., Berglund L., Hertzberg M., Niittylä T, & Oksman K. (2021). The Effect of High Lignin Content on Oxidative Nanofibrillation of Wood Cell Wall. Nanomaterials, 11(5), 1179.

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Western Blot Analysis of Brain Proteins

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Small portions of the snap frozen resected brain tissue from EPI and NON-EPI regions were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) with protease inhibitor (Sigma-Aldrich, USA). The tissue suspension was centrifuged at 14000G (Avanti-J25I, Beckman Coulter, USA), thereafter supernatant was collected and concentration of protein was measured by Bradford Method.
P-gp/MDR1 was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred to PVDF (polyvinylidene fluoride) membranes (EMD Millipore Corp., Billerica, MA, USA) in semi-dry transfer (trans-Biot™ SD, Bio-Rad, USA). All other protein targets were separated by 10% SDS-PAGE. In brief, the membranes were probed overnight with primary antibody (see target proteins listed in Supplemental Table 1a). After incubation with primary antibody the membranes were probed with appropriate secondary antibody (Supplemental Table 1b) as previously described [14 (link)]. For the target proteins either the PVDF membranes were incubated in stripping buffer in 50 °C for 30 minutes followed by blocking of the membranes or a fresh gel was simultaneously repeated with the samples. In each case, the protein expression was normalized by β-actin (as loading control) and quantified by ImageJ software.

Williams S., Hossain M., Ferguson L., Busch R.M., Marchi N., Gonzalez-Martinez J., Perucca E., Najm I.M, & Ghosh C. (2019). Neurovascular drug biotransformation machinery in focal human epilepsies: Brain CYP3A4 correlates with seizure frequency and antiepileptic drug therapy. Molecular neurobiology, 56(12), 8392-8407.

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