The pipAf gene was amplified using the above mentioned template from Müller et. al. 2007 (link). It was cloned in the expression vector pET30 Ek/lic containing an N-terminal His-tag with the help of the ligation independent Ek/lic system according to the protocol proposed by Novagen, UK. E.coli Rosetta DE2 was transformed with the resulting construct pET30/pip. 200 ml of Luria Bertami broth (LB) (Sambrook & Russell, 2006) with kanamycin (50 µg/ml) and chloramphenicol (34 µg/ml) was inoculated with 2 ml of an overnight culture and incubated at 37°C on a rotary shaker (180 rpm). Expression was induced with 1mM IPTG after the culture broth has reached an OD578 of 0.4. The induced culture was further incubated for 18h. The cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, 20 mM imidazol, protease inhibitor (Roche, Mannheim, Germany)) and lysed by 3-4 passages through Emulsiflex B15 (Avestin, Ottawa, Canada). Protein purification was either carried out by gravity flow Ni NTA Superflow columns (Iba) or FPLC using a His-TrapFF 1ml column (GE Healthcare, Munich, Germany). Concentrated protein eluates were stored at 4°C.
Emulsiflex b15
Manufactured by Avestin
Sourced in Canada
The EmulsiFlex-B15 is a high-pressure homogenizer designed for the continuous processing of emulsions, suspensions, and other fluid products. It utilizes a piston-based design to generate high pressures up to 2,000 bar, enabling efficient particle size reduction and homogenization of a wide range of materials.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Histrap ff 1 ml column, by GE Healthcare (2 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Centricon plus 70 centrifugal filter unit, by Merck Group (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Typhoon scanner, by GE Healthcare (1 mentions)
P40st rotor, by Hitachi (1 mentions)
Piston gradient fractionator, by BioComp Instruments (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Temozolomide, by Merck Group (1 mentions)
Q125 sonicator, by Thermo Fisher Scientific (1 mentions)
1 2 dipalmitoyl rac glycero 3 phosphocholine, by Merck Group (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Wise tis homogenizer hg 15d, by Daihan Scientific (1 mentions)
Vcx 750, by Sonics (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Quick start protein assay, by Bio-Rad (1 mentions)
Las 4000 image analyzer, by GE Healthcare (1 mentions)
22 protocols using emulsiflex b15
1
Recombinant PipAf Protein Expression
2
Protein Isolation from Candida albicans Cell Wall
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To isolate proteins from the DTT-soluble cell wall fraction of C. albicans, cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 1x EDTA-free protease inhibitor cocktail (Complete; Roche) 1mM phenylmethyl-sulfonylfluoride) and lysed using bead beating or high-pressure homogenization (Emulsiflex B-15, Avestin). The remaining cell debris was washed once with lysis buffer and then twice in SH-reducing buffer (50 mM Tris-HCl pH 7.5, 100 mM DTT or alternatively 4% ß-mercaptoethanol). Wash fractions were pooled and analyzed directly by SDS-PAGE/Western blotting.
Concentration of culture supernatant was achieved using Amicon Ultra-15 centrifugal filter units, MWCO 10 kDa (Merck) or Centricon® Plus-70 centrifugal filter units (Merck) depending on the supernatant volume. Centrifugation speed and time were chosen according to the specifications of the manufacturer.
Concentration of culture supernatant was achieved using Amicon Ultra-15 centrifugal filter units, MWCO 10 kDa (Merck) or Centricon® Plus-70 centrifugal filter units (Merck) depending on the supernatant volume. Centrifugation speed and time were chosen according to the specifications of the manufacturer.
3
Purification and Analysis of Recombinant Proteins
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E. coli BL-21 pRosettas containing the three expression vectors were cultured in terrific broth with 100 ug/ml ampicillin and 30 ug/ml chloramphenicol overnight then diluted 1:1000 into 50 ml of fresh media. These cultures were grown at 37 C with 250 rpm shaking until reaching an OD600 between 0.9 and 1.0. The cultures were induced with 1 mM IPTG and transferred to a 16 C incubator overnight. The following morning the cells were pelleted by centrifugation at 5,000 × g for 10 minutes and flash frozen in liquid nitrogen.
Frozen cell pellets were resuspended in 10 ml of MQA buffer and lysed in an Emulsiflex-B15 (Avestin). Lysates were cleared by centrifugation at 22,000 × g for 30 minutes. Ammonium sulfate was added to the cleared lysate to a final concentration of 55% saturation. After a 15 minute incubation at 4 C, the insoluble fraction was removed by centrifugation at 22,000 × g for 30 minutes. 2 µl of the lysed, cleared, and ammonium sulfate soluble fractions were loaded onto 15% Tris-glycine SDS Polyacrylamide gels and electrophoresed at 25 mA for 1 hour. Gels were stained with Sypro Ruby (ThermoFisher) and imaged on Typhoon scanner (GE).
Frozen cell pellets were resuspended in 10 ml of MQA buffer and lysed in an Emulsiflex-B15 (Avestin). Lysates were cleared by centrifugation at 22,000 × g for 30 minutes. Ammonium sulfate was added to the cleared lysate to a final concentration of 55% saturation. After a 15 minute incubation at 4 C, the insoluble fraction was removed by centrifugation at 22,000 × g for 30 minutes. 2 µl of the lysed, cleared, and ammonium sulfate soluble fractions were loaded onto 15% Tris-glycine SDS Polyacrylamide gels and electrophoresed at 25 mA for 1 hour. Gels were stained with Sypro Ruby (ThermoFisher) and imaged on Typhoon scanner (GE).
4
Ultracentrifugation of Bacterial Protein Complexes
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Corynebacterium glutamicum cells were lysed in TESG15 buffer (50 mM TES‐NaOH [pH 7.6] and 15% glycerol) containing 1 mM PMSF, 1 mM DTT, 10 µg/ml DNase, and 10 µg/ml RNase at high pressure using EmulsiFlex‐B15 (Avestin Inc., Ottawa). After cell debris removal by centrifugation (7000 g, 10 min, 4°C), the cleared lysate containing 1.2 mg of protein was layered onto a 10%–30% or 15%–45% sucrose density gradient in 50 mM TES‐NaOH (pH 7.6). The gradient was centrifuged at 36,000 rpm (max. 230,000 g) in a HITACHI P40ST rotor for 17 h at 4°C. After centrifugation, the gradient was fractionated using a Piston Gradient Fractionator (BioComP Instruments, Fredericton) into 21 fractions of 0.5 ml and the bottom fraction. Escherichia coli DH5α cells were grown in 2 × YT medium until the OD660 reached 3.5 and processed using the same ultracentrifuge protocol. Aliquots (10 μl) were separated by SDS‐PAGE and subjected to Western blot analysis to detect each subunit of PDH and ODH. Denatured samples were prepared by boiling for 5 min in the presence of 1% SDS before ultracentrifugation.
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Corynebacterium glutamicum cells were lysed in TESG15 buffer (50 mM TES‐NaOH [pH 7.6] and 15% glycerol) containing 1 mM PMSF, 1 mM DTT, 10 µg/ml DNase, and 10 µg/ml RNase at high pressure using EmulsiFlex‐B15 (Avestin Inc., Ottawa). After cell debris removal by centrifugation (7000 g, 10 min, 4°C), the cleared lysate containing 1.2 mg of protein was layered onto a 10%–30% or 15%–45% sucrose density gradient in 50 mM TES‐NaOH (pH 7.6). The gradient was centrifuged at 36,000 rpm (max. 230,000 g) in a HITACHI P40ST rotor for 17 h at 4°C. After centrifugation, the gradient was fractionated using a Piston Gradient Fractionator (BioComP Instruments, Fredericton) into 21 fractions of 0.5 ml and the bottom fraction. Escherichia coli DH5α cells were grown in 2 × YT medium until the OD660 reached 3.5 and processed using the same ultracentrifuge protocol. Aliquots (10 μl) were separated by SDS‐PAGE and subjected to Western blot analysis to detect each subunit of PDH and ODH. Denatured samples were prepared by boiling for 5 min in the presence of 1% SDS before ultracentrifugation.
5
Preparation of PFD Liposomes
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PFD liposomes was prepared by the film hydration method reported by Ng et al19 (link) with slight modifications. Briefly, egg phosphatidyl choline, cholesterol, and PFD (mass ratio 60:7.5:1.5) were dissolved in dichloromethane. A thin film of dry lipid was deposited on the inner wall of the flask by evaporating the solvent under vacuum at 40°C. The film was hydrated at 40°C with phosphate-buffered saline (pH 7.4) followed by sonication for six cycles at 1,200 bar with an ultrasound probe (EmulsiFlex-B15, Avestin, Ottawa, ON, Canada) to form the PFD liposomes. The final suspension volume was about 10 mL, which was then passed through a 1 μm mesh sieve to remove aggregates (recovery rate >85%). The liposomes were collected by filtration of 5 mm mixed cellulose eater membrane.
6
Fabrication of Lipid-Magnetic Nanovesicles
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25 mg of 1-stearoyl-rac-glycerol (Sigma-Aldrich), 2.5 mg of oleic acid (Sigma-Aldrich), 2.5 mg of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (Sigma-Aldrich), 4 mg of mPEG-DSPE5k (Sigma-Aldrich), and 2.5 mg of temozolomide (Sigma-Aldrich) (when TMZ-LMNVs are fabricated), are mixed with 84.5 μl of an ethanol solution of superparamagnetic iron oxide nanoparticles (15 wt%; US Research Nanomaterials Inc.), inside a 6 ml glass vial. Subsequently, the vial is placed inside an ultrasonic bath (Elmasonic S 35w) set at 70 °C in order to melt the lipids and to allow ethanol to evaporate. After ethanol evaporates, 3 ml of a pre-warmed (70 °C) Tween® 80 (Sigma-Aldrich) solution (1.0 wt%) were added to the lipid mixture and immediately sonicated using an ultrasonic homogenizer (Fisherbrand™ Q125 Sonicator) for 15 min (amplitude 30%, 120 W). After the ultrasonic homogenization, the hot mixture is transferred to a high pressure homogenizer (HPH, EmulsiFlex-B15 from Avestin), where the sample is further homogenized by passing it 5 times through the homogenizer at a pressure of 100 000 psi. After the homogenization, the LMNVs are placed for 30 min at 4 °C to allow the lipid-based structures to stabilize. The LMNVs are purified by centrifugation and washing with ultrapure (Mili-Q) water (3 times for 30 min at 4 °C).
7
Nanoparticle-Encapsulated Curcumin and ICG
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BSA-Cur-NPs and ICG-BSA-Cur-NPs were prepared using nanoparticle albumin-bound (nabTM) technology with some adjustments.39 (link) Briefly, aliquots of 50 mg BSA/1.5 mg ICG or 50 mg BSA were dissolved in 5 mL deionized water (DW). Aliquots (2 mg) of Cur was dissolved in 200 μL of a 9:1 solution of chloroform and ethanol. These two solutions were gently mixed and crudely homogenized by using a Wise Tis homogenizer HG-15D (DAIHAN Scientific Co, Seoul, South Korea) at 10,000 rpm and then by passing them through a high-pressure homogenizer (EmulsiFlex-B15 device Avestin, Ottawa, Ontario, Canada) for nine cycles at 20,000 psi. After removal of chloroform by rotary evaporator at 40°C for 15 min under reduced pressure, the resulting NPs were mildly centrifuged at 6000 rpm. The supernatant was collected and purified with Ultra centrifugal filter units (MWCO: 100 kDa, Amicon® Ultra, Millipore) to remove the unbound ICG and Cur, and was then lyophilized and stored at −20°C until required.
8
Cloning and Purification of pipAf Protein
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The pipAf gene was amplified using the above mentioned template from Müller et al., 2007 (link). It was cloned in the expression vector pET30 Ek/lic containing an N-terminal His-tag with the help of the ligation independent Ek/lic system according to the protocol proposed by Novagen, UK. E.coli Rosetta DE3 was transformed with the resulting construct pET30/pip. 200 ml of Luria Bertami broth (LB) (Sambrook & Russell, 2006 ) with kanamycin (50 μg/ml) and chloramphenicol (34 μg/ml) was inoculated with 2 ml of an overnight culture and incubated at 37°C on a rotary shaker (180 rpm). Expression was induced with 1 mM IPTG after the culture broth has reached an OD578 of 0.4. The induced culture was further incubated for 18 h. The cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, 20 mM imidazol, protease inhibitor (Roche, Mannheim, Germany)) and lysed by 3-4 passages through Emulsiflex B15 (Avestin, Ottawa, Canada). Protein purification was either carried out by gravity flow Ni NTA Superflow columns (Iba) or FPLC using a His-TrapFF 1 ml column (GE Healthcare, Munich, Germany). Concentrated protein eluates were stored at 4°C.
9
Determination of Cellular Lipid Content
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Determination of cellular dry weight occurred by pelleting 2 mL samples (12,000 g for 10 min), washing cells with ddH2O and freeze drying at −80 °C for 24 h in pre-weighed microtubes. Cellular total lipid was obtained by extraction with chloroform and methanol by Folch et al. [11 (link)] (adapted). Cell pellets from 12 mL culture were washed with ddH2O twice and disrupted four times by french press (EmulsiFlex®-B15, Avestin) at 2400 bar. A triplicate of 4 mL cell lysate was transferred to glass vials with screw caps and mixed with 6 mL of Folch reagent (2:1 chloroform/methanol) each. After extraction by shaking at 900 rpm and room temperature for 1 h, 1 mL 0.9% NaCl was added to aid phase separation. Samples were vortexed, centrifuged at 1000 g and the chloroform phase was transferred to pre-weighed glass vials. After evaporation of the solvent und a constant stream of dried nitrogen, vials were weighed and lipid content was calculated per dry weight in % g/g.
10
Synthesis and Characterization of Nano-Sized Calli of Rumex roseus
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Calli of RR (cRR) were ground finely by constant trituration using a mortar and pestle. The ground cRR (200 mg) was dispersed in 5 mL of water, resulting in micro-cRR. The micro-cRR was further size-reduced by a probe sonicator at 40 amp for 5 min (VCX 750, Sonics & Materials, USA). It was then subjected 20 times to HPH (EmulsiFlex-B15, Avestin, Canada), resulting in nano-cRR. The resulting suspensions of cRR were analyzed for particle size distribution and zeta potentials using dynamic light scattering (ELSZ-1000, Otsuka Electronics Co, Japan).
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