Doxorubicin and DiR were loaded in PLGA nanoparticles by an emulsion evaporation method36 (link)37 (link). In brief, 4 mg of DOX-HCL was first converted into free base with 0.01 mol/L NaOH solution and then added directly into 2 mL of acetone containing 30 mg PLGA. Next, 2.5 mL mixture of PLGA and the drug was added slowly into 0.01% sodium carboxymethyl cellulose. Followed by the evaporation of the acetone with gentle stirring overnight, the resulting DOX-PLGA nanoparticles were washed by ultrafiltration technique (500 μL, 30k MWCO, Millipore, USA) at 3000 rpm for 25 minutes to remove unloaded doxorubicin. After the final wash, nanoparticles in the upper chamber of the ultrafiltration centrifuge filter were harvested. Unloaded doxorubicin was in the lower chamber. Nanoparticles containing DiR was also prepared similarly.
30k mwco
Manufactured by Merck Group
Sourced in United States, Germany
The 30k MWCO is a lab equipment product designed for molecular separation and purification processes. It functions as a membrane filter with a molecular weight cutoff of 30,000 Daltons, allowing the effective separation of molecules based on their size and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 0, by Merck Group (1 mentions)
Hitrap q hp 5 ml column, by GE Healthcare (1 mentions)
Akta purifier chromatography system, by GE Healthcare (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hitrap deae ff, by GE Healthcare (1 mentions)
4 protocols using 30k mwco
1
PLGA Nanoparticles for Doxorubicin and DiR
2
Fluorescent Protein Conjugation Protocol
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TRITC (1 mg mL−1) in anhydrous DMSO was prepared freshly. 3 mg AF, fetuin, BSA and AGF were dissolved in 1 mL 100 mM carbonate/bicarbonate buffer (pH 9.0), respectively. While stirring, 35 μL of TRITC was slowly added to the protein solution. The mixture was incubated over night at room temperature in the dark. Ultrafiltration (Amicon Ultra-0.5, 30 k MWCO, Millipore) was used to separate and concentrate the TRITC-proteins at 8000 rpm for 10 min. The obtained conjugates were washed more than 6 times with PBS to remove excess and hydrolyzed TRITC. The molar ratios of TRITC to proteins were obtained by determining the absorbance of TRITC at 555 nm with a molar extinction coefficient of 85000 M−1 cm−1 and the absorbance of AF, fetuin, BSA and AGF at 280 nm with the percent extinction coefficients of 4.9, 4.5, 6.67 and 5.19, respectively. Due to the effect of TRITC absorbance at 280 nm, a correction factor of 0.34, which was obtained from the absorbance of TRITC at 280 and 555 nm, was used for determination of protein concentrations. The molar ratios of TRITC to proteins were characterized to be 0.5, 0.3, 1.0 and 0.3, respectively.
3
Purification of IRDIG35563 Protein
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Example 4
IRDIG35563 Purification.
Harvested Pf cells containing IRDIG35563 were sonicated in lysis buffer consisting of 50 mM Tris (pH 8.0), 1 M NaCl, 10% glycerol and 2 mM EDTA with 50 μL of Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Mo.) per 25 mL buffer. The extract was centrifuged at 20,000×g for 40 minutes. The soluble protein in the supernatant was precipitated with 50% ammonium sulfate and centrifuged at 20,000×g for 30 minutes. The pellet was resuspended in 50 mM Tris (pH 8.0) and centrifuged at 20,000×g for 20 minutes to pellet any soluble material. The supernatant containing IRDIG35563 was purified by anion exchange chromatography using a HiTrap™ Q HP 5 mL column with an AKTA Purifier chromatography system (GE Healthcare, UK). The column was equilibrated in 50 mM Tris (pH 8.0), and proteins were eluted with a stepwise gradient to 1 M NaCl. Protein-containing fractions were combined and concentrated using Amicon® Ultra-15 Centrifugal Filter Devices with a 30K MWCO (EMD Millipore, Burlington, Mass.). The IRDIG35563 protein sample was dialyzed overnight against 50 mM Tris (pH 8.), and total protein concentrations were measured with the NanoDrop 2000C Spectrophotometer (Thermo Scientific, Waltham, Mass.), using the A280 method.
4
Phycobiliprotein Purification from A. maxima
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Fresh A. maxima cell pellet was taken and lysed in an equivalent volume of 50 mm potassium phosphate (pH 7.0) using a combination of freeze‐thaw (−80 to +25 °C) cycles and sonication. The lysate was then centrifuged at 16 000 g for 5 min. The blue‐green supernatant was clarified with a stepwise ammonium sulfate (AmSO4) precipitation, collecting the blue supernatant after 25% AmSO4 and blue precipitate after 60% AmSO4 containing the phycobiliproteins. After each step centrifugation was performed at 16 000 g for 15 min. The mixture of phycobiliproteins was further purified using size exclusion chromatography (Superdex Increase 200, 10/300 GL; GE Healthcare, Uppsala, Sweden) into 100 mm ammonium acetate (pH 6.8). Fractions corresponding to hexameric phycobiliproteins were pooled. For higher purity PC, the 60% AmSO4 precipitate was buffer exchanged into 50 mm ammonium acetate (pH 5.0) and PC separated from APC by anion exchange chromatography (HiTrap DEAE FF; GE Healthcare) with a linear gradient to 50 mm ammonium acetate (pH 3.8) [37 ]. For all purifications, fractions were further exchanged into 100 mm ammonium acetate (pH 6.8) with an Amicon Ultra 0.5 mL centrifugal concentrator with a 30k MWCO (Merck Millipore, Darmstadt, Germany). All phycobiliproteins were stored at 4 °C in the dark to prevent degradation of the bilins and protein complexes.
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