Emulsiflex c5

Manufactured by Avestin

Sourced in Canada, Germany, United States

The EmulsiFlex-C5 is a high-pressure homogenizer designed for the production of emulsions, suspensions, and liposomes. It utilizes a high-pressure pumping system to create controlled disruptive forces that reduce particle size and enhance dispersion. The EmulsiFlex-C5 is a compact, benchtop unit suitable for use in laboratory settings.

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Lab products found in correlation

Ni nta agarose, by Qiagen (7 mentions) Hitrap, by Cytiva (5 mentions) Ni nta agarose resin, by Qiagen (4 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Superdex 75 column, by GE Healthcare (3 mentions) Superdex 200 gel filtration column, by Cytiva (3 mentions) Source 15q resin, by Cytiva (3 mentions) Ni nta superflow resin, by Qiagen (3 mentions) Histrap ff crude, by GE Healthcare (3 mentions) Xk16 column, by GE Healthcare (3 mentions) Whatman, by Cytiva (2 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (2 mentions) Superdex 200, by GE Healthcare (2 mentions) Hiprep, by Cytiva (2 mentions) Thrombin, by GE Healthcare (2 mentions) Christ alpha 1 3 ld plus, by Martin Christ (2 mentions) Ultra 10 k concentrators, by GE Healthcare (2 mentions) Source 15s resin, by Cytiva (2 mentions) Superdex 200 prepgrade column, by Cytiva (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions)

Spelling variants of this product

EmulsiFlex-C5 homogenizer (50 citations) EmulsiFlex (27 citations) Emulsiflex C5 high-pressure homogenizer (15 citations) EmulsiFlex-C5 cell homogenizer (2 citations) EmulsiFlex-C5 cell disrupter (2 citations) Emulsiflex C5 high pressure homogeniser (2 citations) EmulsiFlex-C5 high (2 citations) EmulsiFlex-C5 cell disruptor (2 citations)

106 protocols using emulsiflex c5

Glutathione-Coated Nanosuspension for Resveratrol Delivery

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Before performing drug loading, nanosuspension of GSH-NSs (10 mg/mL in water or saline) was prepared by high shear homogenizer (Ultraturrax^®, IKA, Konigswinter, Germany) for 10-15 min at 24,000 rpm followed by high-pressure homogenization (HPH) for 1.5 hours at a back pressure of 500 bar using an EmulsiFlex C5 instrument (EmulsiFlex C5, Avestin, USA). Later, nanosuspension was dialyzed for a few minutes.
RV-loaded GSH-NSs were prepared by adding RV in a different weight ratio of 1:2, 1:4, and, 1:6 (w/w; drug: nanosponge) in a nanosuspension of GSH-NSs (10 mg/mL). Later, samples were sonicated for 20 minutes followed by continuous stirring for 24 hours in dark. Samples were subjected to mild centrifugation and supernatant was collected followed by dialysis in water for a few minutes to remove the unloaded drug. RV-loaded GSH-NSs were freeze-dried and stored in a desiccator for further characterization. Fluorescent NSs were prepared in a similar manner by taking NS suspension (10 mg/mL) in saline with 0.1 mg/mL coumarin-6 (C-6).

Palminteri M., Dhakar N.K., Ferraresi A., Caldera F., Vidoni C., Trotta F, & Isidoro C. (2021). Cyclodextrin nanosponge for the GSH-mediated delivery of Resveratrol in human cancer cells. Nanotheranostics, 5(2), 197-212.

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Purification of recombinant HP0860 protein

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The transformed BL21 (DE3)/pET28a-HP0860 cells were grown in 2× YT medium containing 50 μg/mL kanamycin with shaking. When OD₆₀₀ reached 0.6–0.8, the transformed cells were induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG; Sigma-Aldrich; I6758) for 4 hours at 37°C. The IPTG-induced cells were harvested by centrifugation (4000

\times

g for 20 minutes at 4°C), and then the cell pellet was suspended in 30 mL of homogenization buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 20 mM imidazole) and passed through a high-pressure homogenizer (EmulsiFlex-C5; AVESTIN; Canada) to break cells. The obtained lysate was centrifuged (11,000

\times

g, 20 minutes at 4°C), and the resulting supernatant was applied for affinity purification with IMAC Sepharose 6 FastFlow resins (GE Healthcare; 17–0921-08) and 100 mM CoCl₂ as the immobilized metal ion according to manufacturer’s recommendation. After thoroughly washing, the protein was eluted with a buffer containing 250 mM imidazole. The desired eluted fractions were combined and concentrated by using an Amicon Ultra 15 concentrator (Millipore; Z717185) to obtain recombinant HP0860 protein.

Chiu S.F., Teng K.W., Wang P.C., Chung H.Y., Wang C.J., Cheng H.C, & Kao M.C. (2021). Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence. Virulence, 12(1), 1610-1628.

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Nanomerics MET and MPA Formulation

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Nanomerics MET (60 mg) was dispersed in glycerol (2.75%, 8 mL) at a concentration of 0.75% w/v. To this was added MPA (8mg) to give a GCPQ, MPA weight ratio of 7.5: 1 and the mixture was initially vortexed, stirred for 2h and subsequently processed for 15 cycles at 18,000 psi using a high pressure homogeniser (Avestin Emulsiflex C5, Avestin, Germany).
After high pressure homogenisation, the formulation was transferred to glass vials. The pH was adjusted to pH = 7.4 using NaOH (1M), a magnetic stirrer and a pH meter (Jenway 3310, VWR, UK). The resulting solution was filtered using a 2mL syringe (BD Plastipak, LOT: 1503003) and 0.22μm Millex GP filter unit (Millipore, LOT: R7PA99493).
A blank formulation is prepared by carrying out the steps above but omitting the addition of MPA. Spiked formulations used in precision tests were prepared by spiking a portion of the blank formulation with an equal volume of MPA (of a required concentration) dissolved in acetonitrile.

Al-Kulabi A., Gooden L, & Uchegbu I.F. (2021). Nanoparticulate Mycophenolic Acid Eye Drops - Analytical Validation of a High Performance Liquid Chromatography Assay and Stability Studies. Pharmaceutical nanotechnology, 9(2).

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Production of Labeled Protein via Inclusion Body Solubilization

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The coding sequence for the first 178 amino acids of pOA were amplified by PCR from a parent pOA plasmid using primers for Gibson assembly that added a GSGS linker at the C-terminus. The entire mDHFR ORF was PCR amplified from a mDHFR plasmid with a C-terminal AviTag fused to the mDHFR fragment using Gibson Assembly (NEB). The backbone contained a T7 promoter. Cysteines were added at the desired positions using either site directed mutagenesis or insertion of synthetic DNA fragments (gBlocks, IDT). The p86 peptide sequence VSGWRLFKKIS^{34 (link)} was added to the end of the of the construct via PCR. The resulting plasmid (termed pOA-mDHFR) was transformed into E. coli strain MM52, which contains temperature sensitive SecA allele. A starter culture was grown at the permissive temperature (30 °C) until it reached OD₆₀₀ of 0.5. The culture was then shifted to the restrictive temperature (37 °C), and expression was induced with 1 mM IPTG for 4 h. The cell pellet was harvested and washed with cold 1X PBS. Cells were lysed using an EmulsiFlex-C5 (Avestin) in 1X PBS pH 7.5. Inclusion bodies were washed with 1X PBS three times. Inclusion bodies were pelleted and frozen in liquid N₂. The substrate protein was solubilized from inclusion bodies in 6 M urea before use.

Gupta R., Toptygin D, & Kaiser C.M. (2020). The SecA motor generates mechanical force during protein translocation. Nature Communications, 11, 3802.

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Scalable Liposome Preparation by Cross-Flow Injection

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Larger quantities of liposomes (up to 200 mL preparation volume) were prepared using a scalable cross-flow injection setup [59 ,60 (link)]. Briefly, an ethanolic lipid solution was mixed with buffer (with or without T helper cell peptide) using a proprietary mixing module. The preparation was performed in a heating cabinet at up to 60 °C. Downsizing by means of extrusion was performed at room temperature on an EmulsiFlex-C5 (Avestin Inc., Ottawa, ON, Canada) equipped with 100 nm Whatman^® Nuclepore track-etched membranes (GE Healthcare UK Limited, Buckinghamshire, UK).

Suleiman E., Mayer J., Lehner E., Kohlhauser B., Katholnig A., Batzoni M., Damm D., Temchura V., Wagner A., Überla K, & Vorauer-Uhl K. (2020). Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations. Pharmaceutics, 12(10), 979.

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Yeast Cell Fractionation by Gel Filtration

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Gel filtration experiments were performed in a Superose 6 10/300 GL column (GE Healthcare) using an ÄKTAFPLC system. Yeast-state or hyphae-state cells were collected and washed once with phosphate-buffered saline (PBS) and then re-suspended in lysis buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail). The cells were homogenized by a high-pressure homogenizer (EmulsiFlex-C5, Avestin, Inc., Ottawa, Canada), then were centrifuged at 13,000 r.p.m. for 20 min. The supernatant was filtrated with a 0.22 μm membrane, 500 μL of WCEs (5 mg total protein) were injected into Superose 6 column, eluted at a speed of 300 μL/min. The eluted fractions were collected and concentrated by centrifugal filter devices (Amicon Ultra-4, 10 K, Millipore) and further analyzed by western blotting.

Wang X., Zhu W., Chang P., Wu H., Liu H, & Chen J. (2018). Merge and separation of NuA4 and SWR1 complexes control cell fate plasticity in Candida albicans. Cell Discovery, 4, 45.

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Detailed Protocol for CYP Expression

Cited 3 times

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CYPs preparations were carried out essentially as previously described (Swaminathan et al. 2009 (link); Wang et al. 2012a (link); Wang et al. 2011 (link); Wang et al. 2012b (link); Wu et al. 2011 (link); Wu et al. 2013 (link)). Briefly, the CYPs were expressed with AtCPR1 using one of the various CYP expression vectors described above, which were generally co-transformed with the compatible pCDF-Duet1/AtCPR1 vector, into the C43 OverExpress strain of E. coli (Lucigen). Expression cultures were grown in TB medium (pH 7.5, 4 × 100 mL in 250 mL Fernbach flasks) containing appropriate antibiotics. Upon reaching an A₆₀₀ of ~0.8 the cultures were shifted to 16 °C, supplemented with 5 mg/L riboflavin and 75 mg/L 5-aminolevulinic acid, and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 72 hours the cells were harvested via centrifugation (10 min. × 5000 g), resuspended in 30 mL buffer (0.1 M Tris/HCl, pH 7.5, 0.5 mM EDTA, 20% glycerol), and twice passed through a French press homogenizer (Emulsiflex-C5: Avestin) at 1500 psi. The resulting lysates were clarified via centrifugation (20 min. × 14000 g) and the concentration of CYP in the resulting supernatant (cell-free lysate) was quantified by carbon monoxide (CO)-binding difference spectra using the standard extinction coefficient of 91 mM⁻¹cm⁻¹ (Omura and Sato 1964 (link)).

Kitaoka N., Wu Y., Xu M, & Peters R.J. (2015). Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis. Applied microbiology and biotechnology, 99(18), 7549-7558.

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Purification of His-Tagged Proteins from E. coli

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Purified proteins were obtained by growing E. coli BL21(DE3) cells containing a pET28a-based plasmid encoding the desired
protein or Rosetta (DE3) cells containing a pTriEx-3-based plasmid
in 200 mL of Luria–Bertani (LB) medium at 37 °C. Expression
was induced with 0.1 mM IPTG when the culture density (Abs₆₀₀) reached 0.6–0.8 and growth continued for 6 h at 30 °C.
Cultures were harvested by centrifugation at 4000g for 30 min at 4 °C. Cell pellets were stored at −20
°C overnight. Thawed pellets were resuspended in 10 mL equilibration
buffer (25 mM Tris-HCl, pH 7.4, 500 mM NaCl and 20 mM imidazole) and
lysed with a high-pressure homogenizer (Avestin Emulsi-Flex C5). The
insoluble fraction was cleared by centrifugation at 12000g for 30 min at 4 °C. His-tagged protein was purified by gravity
flow using 500 μL HisPur Ni-NTA resin (ThermoFisher). The soluble
fraction was passed through the resin, after which the resin was washed
with 3 mL of wash buffer (25 mM Tris-HCl, pH 7.4, 500 mM NaCl and
50 mM imidazole). Protein was eluted with 1.5 mL elution buffer (25
mM Tris-HCl, pH 7.4, 500 mM NaCl, and 250 mM imidazole). Purified
fractions were desalted and concentrated (Pierce PES Protein Concentrators).

Ludwicki M.B., Li J., Stephens E.A., Roberts R.W., Koide S., Hammond P.T, & DeLisa M.P. (2019). Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic. ACS Central Science, 5(5), 852-866.

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Purification of Recombinant Proteins

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Basic chemicals were obtained from Sigma Chemical, Inc., St. Louis, MO, USA, while Complete^® EDTA-free protease inhibitor cocktail tablets were obtained from Roche Applied Science, Indianapolis, IN, USA and Imidazole was obtained from EMD Biosciences, San Diego, CA, USA. Conical centrifuge tubes were obtained from Corning Life Sciences (Corning, NY, USA), and Amicon Ultra centrifugal filters (Ultracel-50k) were obtained from Millipore, Billerica, MA, USA. Homogenization was performed with a high-pressure homogenizer (EmulsiFlex-C5, Avestin, Inc., Ottawa, Ontario, Canada). HISTRAP FF, HIPREP 26/10 Desalting columns, Superdex™ 200, 10/300GL gel filtration column, and AKTA Purifier UPC 10 were from GE Healthcare Bio-Sciences Corporation (Piscataway, NJ, USA). The Uno S6 column was obtained from Bio-Rad Company (Hercules, CA, USA).

Wu H., Hung R.J, & Terman J.R. (2016). A Simple and Efficient Method for Generating High-quality Recombinant Mical Enzyme for In Vitro Assays. Protein expression and purification, 127, 116-124.

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Purification of KIF1C Motor Domain

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KIF1C motor domain (residues 1–349) was fused to murine KIF5C residues 329–334 followed by 6×His tag (Nitta et al., 2004 (link)) and purified based on Romberg et al. (1998) (link). After expression in Rosetta 2 cells using 0.5 mM isopropyl B-p-thiogalactopyranside for 16 hr at 16°C, cells were suspended in Buffer B (50 mM NaPO₄, pH 7.4, 15 mM imidazole, 250 mM NaCl, 1 mM MgCl₂, 25 µM ATP, protease inhibitors) and disrupted by Emulsiflex C-5 (Avestin, Ottawa, ON). Clarified lysate was incubated with Ni-NTA (Qiagen, Santa Clarita, CA) for 1.5 hr at 4°C and after washing with Buffer B was eluted with Buffer B + 200 mM imidazole. The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl₂, 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs.

Lee P.L., Ohlson M.B, & Pfeffer S.R. (2015). The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization. eLife, 4, e06029.

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