GLFG liposomes were prepared using the thin film hydration method. PEG-GLFG, DOTAP, Cholesterol, and DPPC were prepared at various molar ratios as shown Table 1 . All lipid mixtures were dissolved in EtOH/chloroform (v/v, 1:1) solvent, which was dried using N2 gas and under vacuum for 20 min. Lipid films were hydrated in 5 mM HEPES buffer and sonicated for 30 min. Dox encapsulated liposomes were prepared by the same procedure as mentioned above. Then, 2 mg lipids and 0.5 mg Dox were prepared in EtOH/chloroform solvent in a glass bottle, dried using N2 gas, and further dried under vacuum for 20 min. Thin film were hydrated in 5 mM HEPES and Dox solution. Dox/liposomes were purified using size exclusion chromatography (SEC) (NAP-25, GE Healthcare Co., Chicago, IL, USA). PBS buffer (pH 7.4) was used as a mobile phase at a flow rate of 1 mL/min. Each sample’s absorbance was measured at 480 nm. Drug encapsulation efficiency was calculated as the following Equation (1):
Nap 25
Manufactured by GE Healthcare
Sourced in United States
The NAP-25 is a centrifuge product offered by GE Healthcare. It is designed to separate biological samples through centrifugal force, a core function of laboratory equipment used in various research and diagnostic applications.
Automatically generated - may contain errors
6 protocols using nap 25
1
Doxorubicin-Loaded GLFG Liposomes
2
Isothermal Titration Calorimetry of SBT Binding
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SBT binding was investigated using isothermal titration calorimetry (ITC). The experiments were carried out on purified samples of protein exchanged into 25 mM phosphate buffer containing 25 mM NaCl using a NAP25 desalting column (GE Healthcare). The SBT (supplied as neat liquid) was diluted in identical buffer. Control experiments whereby the SBT was titrated into buffer and buffer into protein exhibited no detectable heat exchange, confirming that there was appropriate match of buffer conditions with no evidence of dilution effects. To maintain consistency between titrations the same stock buffer was used in all protein and SBT preparations. Experiments were conducted at 25°C with an ITC200 (GE Healthcare) with a 40 µl syringe volume and 200 µl cell capacity. Titrations were carried out using between 40 µM and 100 µM of protein in the cell and a tenfold concentration of SBT (between 400 µM and 1 mM). SBT was added into the cell in sequential 1 µl injections (at a rate of 0.5 µl per second) with a 180 second interval between each injection. One site (three parameters) curve fitting was carried out using the MicroCal-supported ITC module within Origin version 7.
3
Dil-Encapsulated Pluronic F127 Micelles
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Example 6
Into an Eppendorf tube was combined a DMSO aliquot (1 μL of a 25 mM solution) of the hydrophobic dye Dil Stain (1,1′-dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine perchlorate (aka—‘Dil’; DilC18(3)) and an aliquot (1 mL) of a Pluronic F127 solution (1% w/v). The resulting solution (final dye concentration=25 μM) was allowed to shake for 4 h on a benchtop shaker to form the Dil-encapsulated Pluronic F127 micelles, which were purified from the free dye by size-exclusion chromatography (NAP 25, GE Healthcare, Arlington Heights, Ill., USA).
The purified Dil-encapsulated micelles were further incubated overnight with lipid-functionalized DBCO-BHQ-2 dT-(NH2)5-T20 sequence to form the Dil-encapsulated, BHQ-2-labeled micellar SNA, which was then isolated following the functionalization and purification protocols discussed above. The quenching of the Dil dye due to the proximity of BHQ-2-labeled DNA with the Pluronic F127 template in this latter sample was confirmed by the decrease of a fluorescence from the Dil dye in the core.
4
Fluorescent Antibody Labeling Protocol
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To examine the intracellular delivery proof-of-concept by fluorescence microscopy, the monoclonal antibody TuBB-9 was labeled with the fluorescent dye fluorescein isothiocyanate (FITC). The antibody is diluted 1:5 in a sodium carbonate buffer consisting of 160 nM Na2CO3 and 333 nM NaHCO3 with pH 9.3 to obtain an antibody concentration of 1 mg/mL. The suspension is centrifuged for 20 min at 1855× g, and the residues remaining in the filter are taken into buffer solution (pH 9.3). Subsequently, 1 mg/mL of FITC (dissolved in DMSO) is added to the TuBB-9 solution. The resulting suspension is incubated at room temperature with constant agitation for 2 h to obtain FITC binding to TuBB-9.
Using a Sephadex column (NAP-25, GE Healthcare Life Sciences, Chicago, IL, USA), which has been buffered with tris-buffered saline (TBS) (pH 8.2), the obtained sample is purified. The eluate is again centrifuged. The sample was removed from the filter, and to wash out any remaining residue in the filter, it was rinsed twice with 500 µL TBS (pH 7.5), and then addition was carried out to the sample. Finally, the concentration of the antibody and the fluorescent dye is determined using an absorption spectrum.
Using a Sephadex column (NAP-25, GE Healthcare Life Sciences, Chicago, IL, USA), which has been buffered with tris-buffered saline (TBS) (pH 8.2), the obtained sample is purified. The eluate is again centrifuged. The sample was removed from the filter, and to wash out any remaining residue in the filter, it was rinsed twice with 500 µL TBS (pH 7.5), and then addition was carried out to the sample. Finally, the concentration of the antibody and the fluorescent dye is determined using an absorption spectrum.
5
Fluorescently Labeled RNA Protocol
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For fluorophone labeling, 10 nmol of each RNA were dissolved in 150 µL borate-buffer (0.1 M sodium tetraborate (Merck), pH 8.4). GFP_1, PSD95_1, CAMK2_1 and β-actin_1 were incubated with 200 nmol ATTO565 NHS (ATTO-TEC), dissolved in 50 µL DMF (Lumiprobe, labeling grade), for 4 h at 37 °C. PSD95_2, CAMK2_2 and Beta Actin_2 were incubated with 200 nmol ATTO647N NHS (ATTO-TEC), dissolved in 50 µL DMF, for 4 h at 37 °C. Buffer and the excess of fluorophore were removed by size-exclusion chromatography (NAP 25, GE Healthcare). The solvent was evaporated at 4 °C using a vacuum concentrator. The residue was purified by RP-HPLC on an Agilent 1200 equipped with an Xbridge BEH C18 OBD (300 Å, 3.5 µm, 4.6 × 250 mm, 1 mL/min, 60 °C). As solvents 400 mM hexafluoroisopropanol, 16.3 mM Et3N, pH 8.3 and MeOH were used with a gradient from 5% MeOH to 100% MeOH in 50 min.
6
Melamine Detection in Milk via SERS
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The milk samples with 0.5 % fat content (Dansk Maelk, Jacobs Douwe Egberts Professional, København, Denmark) were purchased from a local commercial supplier. The samples, spiked with melamine, were pretreated prior to SERS measurement, using a gel filtration column (NAP-25, GE Healthcare, Pittsburgh, PA, USA) as also described by A. Kim et al. 61 (link) In short, 2.5 ml milk samples, spiked with various melamine concentrations (0.3 ppm -2.5 ppm) using the pH 7.4 stock solutions, were loaded into the saturated filtration column. The milk was allowed to sink into the column and subsequently eluted in 2.5 ml fractions with PBS pH 7.4. For SERS measurements the pH of the fractions were adjusted with 10 % HCl to a pH value between 3 to 4, which was verified with pH paper (Merck KGaA, Darmstadt, Germany).
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