Human IgG was diluted in 0.01N HCl to make up IgG concentration of 1 mg/mL (ie, 10 mg) in 10 mL of 0.01 N HCl. A total of 0.73 mg of miRNA-29b was then added. The mixture was stirred on a magnetic stirrer until all the components were fully dissolved. The mixture was titrated with 0.01 N NaOH up to a pH value close to 7. Nanoparticles were spontaneously formed at the pH value very close to 7. The nanoparticles were allowed to mix on the magnetic stirrer for ~10 minutes. The colloidal suspension was centrifuged using a microcentrifuge (Eppendorf centrifuge 5418) at 2,000 rpm for 5 minutes. The supernatant was either decanted or kept for the measurement of unencapsulated miRNA-29b to be used to calculate encapsulation efficiency and loading capacity. Nanoparticles were then rinsed three times with double distilled deionized water. Nanoparticles were subsequently suspended in 0.2% v/v poloxamer-188, with gentle shaking for 10 minutes in order to coat nanoparticles with poloxamer-188. Nanoparticles were centrifuged and the supernatant decanted before being rinsed thrice with double distilled deionized water. Particles were then loaded into a freeze dryer (Labconco Freeze Zone 4.6), and lyophilization was performed for 48 hours.
Freezezone 4
Manufactured by Labconco
Sourced in United States
The FreezeZone 4.5 is a benchtop freeze dryer designed for laboratory use. It has a condenser capacity of 4.5 liters and can accommodate various sample sizes and configurations.
Automatically generated - may contain errors
Lab products found in correlation
Centrifuge 5418, by Eppendorf (3 mentions)
Delta 5 plus, by Thermo Fisher Scientific (1 mentions)
Conflo 4, by Thermo Fisher Scientific (1 mentions)
Whatman number 1 paper, by Cytiva (1 mentions)
Heating bath b 490, by Büchi (1 mentions)
Whatman, by Cytiva (1 mentions)
Lm 3100, by PerkinElmer (1 mentions)
Ultrasonic homogenizer 4710, by Cole-Parmer (1 mentions)
L7 55, by Beckman Coulter (1 mentions)
7 protocols using freezezone 4
1
IgG-miRNA-29b Nanoparticle Formulation
2
Enzalutamide-Loaded Hybrid Nanoparticles
3
Nanoparticle Formulation for siRNA Delivery
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50 mg of excipient-free human IgG was dissolved in 0.01 N HCl containing 20 mg of poloxamer-188. 187 μg of siRNA was then added to make a 10-mL total solution in a 50-mL beaker. The final concentration of human IgG in the solution amounted to 5 mg/mL. This solution was then slowly titrated with 0.01 N NaOH to bring the pH of the mixture to 7, which is the isoelectric point (pI) of human IgG as determined in our laboratory using isoelectric focusing. The nanoparticles were continuously stirred on a magnetic stirrer for 10 min. To isolate the nanoparticles from the precipitation medium, the suspension was centrifuged with a microcentrifuge (Eppendorf centrifuge 5418) at 2,000 rpm for 5 min. Nanoparticles were then rinsed with double-distilled deionized water before being redispersed in water and snap-frozen using liquid nitrogen. This was then loaded into a freeze dryer (Labconco FreezeZone 4.6), and lyophilization was performed for 48 hr.
4
Measuring Kelp Nutrient Status via Isotopes
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Stable isotope ratios were measured to provide evidence of inorganic carbon uptake strategies and C and N content were measured to provide indications on the nutrient status of the kelp. Samples for determination of δ13C isotopic ratios and carbon and nitrogen content were destructively sampled and frozen at –20°C until all treatments had been measured. Following this, all samples were freeze dried (FreezeZone 4.5, Labconco) and kept at –20°C until later analysis. δ13C isotopic ratios and carbon and nitrogen content were determined by weighing approximately 5 mg of dried tissue into tin cups (Sercon, UK) and analysed using an elemental analyser (NA1500, Fisons Instruments) coupled to an isotope ratio mass spectrometer (Delta V Plus, ThermoFisher Scientific) via a Universal Continuous Flow Interface (Conflo IV, ThermoFisher Scientific). Combustion and reduction were achieved at 1020°C and 650°C, respectively. Isotope values were normalized to the Vienna Pee Dee Belemnite scale via a three-point calibration using certified reference material and both precision and accuracy were ±0.1% (1 s.d.).
5
Phytochemical Extraction and Characterization
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Stem, leaf, root, and whole-plant fractions were subjected to drying in a vacuum oven for 24 h, which was conducted at 45°C except in the case of the roots and stems, which were dried at 60°C. The dried materials were pulverized and passed through a Number 20 sieve (WS Tyler). A 35 g aliquot of the sieved sample was then mixed with a solution of ethanol and 5% acetic acid (95 : 5 ratio), and maceration was carried out via constant stirring for 72 h on a stir plate in complete darkness at room temperature. The sample was subsequently vacuum filtered through Whatman Number 1 paper, and the residue was extracted to exhaustion with the acid-ethanol mixture via sonication for 20 min (Branson 3510). This sample was filtered, and the two solvents were mixed and evaporated using a rotatory evaporator (Buchi Heating Bath B-490, Buchi Rotvapor R-200). Finally, the extract was lyophilized for 48 h (Freeze zone 4.5, Labconco), and the dried extracts were maintained at −20°C for subsequent analysis. All the extractions were performed in triplicate and the glycoalkaloids, total phenolic, flavonoids, chlorophyll, antioxidant, and antibacterial activity were measured.
6
Chickpea Preparation and Analysis
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The chickpea grains were cooked by the method of [66 (link)], with some modifications. The grains (500 g) were soaked in 2.5 L of water for 22 h and, after that, the grains were cooked by steaming for 60 min. During soaking, the water absorption and texture were determined as described below. The raw and cooked grains were analyzed in their proximal analysis and color. The controlled atmospheres study and digestibility were determined only in cooked samples. For the phenolic extractions (free, conjugated, and bound) and the antioxidant capacity analysis, the raw and cooked samples were lyophilized (Freeze Zone 4.5, Labconco, Kansas city, MO, USA) and milled in a Perten laboratory mill model LM3100 (PerkinElmer, Waltham, MA, USA) to a final particle size of 0.5 mm mesh.
7
Bacillus thuringiensis Spore Isolation
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Ls 2362 was grown in MBS broth25 (link), and Bti strains 4Q5, 4Q7/pWF45, 4Q7/pBU-cyt1Aa-binA, and 4Q7/p45S1 were grown in 50 ml of NBG13 (link), 14 (link) appropriately supplemented with 25 μg/ml erythromycin and 3 μg/ml tetracycline, at 28 °C for 4 days by which time >95% of the cells had sporulated and lysed. Spores and crystals were collected by centrifugation at 6,500 g for 15 min, washed 2x in double-distilled (dd) H2O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash, and lyophilized (FreezeZone 4.5, Labconco) for storage.
To isolate parasporal bodies, spore/parasporal body mixtures collected from 50 ml cultures were resuspended in 15 ml ddH2O and sonicated twice at 50% duty cycle for 15 s using the Ultrasonic Homogenizer 4710 (Cole-Parmer Instrument Co.). Five-milliliter samples were loaded onto a sucrose gradient cushion (30–65% w/v), which was then centrifuged at 20,000 g for 45 min at 20 °C in a Beckman L7–55 ultracentrifuge using the SW28 rotor. Bands containing parasporal bodies were collected and washed twice in ddH2O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash and lyophilized for storage.
To isolate parasporal bodies, spore/parasporal body mixtures collected from 50 ml cultures were resuspended in 15 ml ddH2O and sonicated twice at 50% duty cycle for 15 s using the Ultrasonic Homogenizer 4710 (Cole-Parmer Instrument Co.). Five-milliliter samples were loaded onto a sucrose gradient cushion (30–65% w/v), which was then centrifuged at 20,000 g for 45 min at 20 °C in a Beckman L7–55 ultracentrifuge using the SW28 rotor. Bands containing parasporal bodies were collected and washed twice in ddH2O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash and lyophilized for storage.
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