Nisin encapsulation efficiency was measured via a Bradford assay. A half milliliter of SLN or SLN-Nisin was placed in a 100 kDa spin column (Millipore, Ireland) and centrifuged at 10,000 RPM for 5 minutes to separate free nisin from the incorporated nisin. The filtrate was, then, collected and stored. Next, 500 μL of a 1% SDS (Sigma–Aldrich, USA) solution was added to the column to disrupt the nanoparticles and release the incorporated nisin. This second filtrate was collected and stored. Finally, the total protein content from both filtrates (i.e., either free or incorporated nisin) was quantified using a Pierce™ BCA Protein Assay Kit (Thermo-Fisher Scientific, USA) according to the manufacturer’s instructions in a Spectramax M2 microplate reader (Molecular Devices, USA).
100 kda spin column
Manufactured by Merck Group
Sourced in Ireland, United States
The 100 kDa spin column is a laboratory filtration device used to separate and concentrate macromolecules based on their molecular weight. It is designed to retain molecules larger than 100 kilodaltons (kDa) while allowing smaller molecules to pass through. This spin column can be used to purify and concentrate proteins, DNA, RNA, and other high-molecular-weight compounds from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g, by Addgene (4 mentions)
Pspax, by Addgene (4 mentions)
Flow cytometry, by Beckman Coulter (2 mentions)
Ow cytometry, by Beckman Coulter (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Formalin, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
7 protocols using 100 kda spin column
1
Measuring Nisin Encapsulation Efficiency
2
Lentiviral CRISPR Knockout Generation
3
Lentiviral Vector Production for FGF21 and GLP1
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Lentiviral vector plasmids and packaging plasmids (psPAX and pMD.2G) were purchased from Addgene. Lentiviral particles carrying pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1, and pCDH-EF1-GLP1 were produced through the transfection of HEK293T (ATCC) packaging cells with a 3rd generation plasmid system. HEK293T cells were transfected with 24 μg of plasmids, 48 μl of Lipofectamine LTX, and 24 μl of PLUS reagents, and the proportions of the pMD.2G, psPAX, and pCDH-EF1 plasmids were 1:2:3. The supernatants were collected at 24 and 48 h after transfection, filtered through 0.45-μm filters, and harvested by ultrafiltration with a 100-kDa spin column (Millipore) at 4 °C and 4000 g for 30 min. The lentiviral particles were aliquoted and stored at − 80 °C until use. The transfection efficiency was determined based on EGFP expression using flow cytometry (Beckman), and the viral titers were determined according to the following equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/volume of added virus fluid (mL).
4
Inactivation and Concentration of Viruses
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Ten plates of MDCK cells (2 × 107/plate) incubated in 20 mL of MEM-α with 0.5 µg/mL TPCK-trypsin were infected with viruses at MOI = 0.001. After 72 h of incubation at 37 °C, 200 mL of virus-containing culture supernatant was collected, centrifuged at 3000× g at 4 °C for 5 min to remove cell debris, and inactivated with 0.01% formalin (Sigma) at 4 °C for 24 h. The inactivated virus-containing solution was then concentrated to 30 mL using a 100 kDa spin column (Millipore, Burlington, MA, USA). Concentrated viruses were overlaid onto 5 mL of a 20% sucrose solution (w/v) dissolved in TNE buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 10 mM, pH 7.4) in six ultracentrifugation tubes (Hitachi, Tokyo, Japan). After ultracentrifugation at 82,700× g and 4 °C for 2 h, the inactivated virus pellets were dissolved in 600 µL of PBS and stored at −80 °C [23 (link),24 (link)]. The total protein concentration of the inactivated viruses was measured using the Bradford protein assay (Bio-Rad, Hercules, MA, USA).
5
Lentiviral-Mediated Transgene Delivery
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Lentiviral vector plasmids and packaging plasmids (psPAX and pMD.2G) were purchased from Addgene.
Lentiviral particles carrying pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1 and pCDH-EF1-GLP1 were produced through the transfection of HEK293T packaging cells with a 3rd generation plasmid system. HEK293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of the pMD.2G, psPAX, and pCDH-EF1 plasmids were 1:2:3. The supernatants were collected at 24 and 48 h after transfection, ltered through 0.45-μm lters, and harvested by ultra ltration with a 100-kDa spin column (Millipore) at 4 °C and 4,000 g for 30 min. The lentiviral particles were aliquoted and stored at -80 °C until use. The transfection e ciency was determined based on EGFP expression using ow cytometry (Beckman), and the viral titers were determined according to the following equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/ volume of added virus uid (mL).
Lentiviral particles carrying pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1 and pCDH-EF1-GLP1 were produced through the transfection of HEK293T packaging cells with a 3rd generation plasmid system. HEK293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of the pMD.2G, psPAX, and pCDH-EF1 plasmids were 1:2:3. The supernatants were collected at 24 and 48 h after transfection, ltered through 0.45-μm lters, and harvested by ultra ltration with a 100-kDa spin column (Millipore) at 4 °C and 4,000 g for 30 min. The lentiviral particles were aliquoted and stored at -80 °C until use. The transfection e ciency was determined based on EGFP expression using ow cytometry (Beckman), and the viral titers were determined according to the following equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/ volume of added virus uid (mL).
6
Lentiviral Vector Production Protocol
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Lentiviral vector plasmids and the packaging plasmids (psPAX and pMD.2G) were purchased from Addgene. Lentiviral particles with pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1 and pCDH-EF1-GLP1 produced through transfection of HEK293T packaging cells with 3rd generation plasmids system. 293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of pMD. 2G, psPAX, and pCDH-EF1 plasmid were 1:2:3. The supernatants were collected at 24 and 48h after transfection, filtered by 0.45 μm filters, and harvested by ultrafiltration with 100 kDa spin column (Millipore) at 4°C and 4,000g for 30 min. Lentiviral particles were dispensed in aliquots, and stored at -80 °C until use. Transfection efficiency was examined by EGFP using flow cytometry (Beckman), and viral titer was determined according to the equation: virus titer (pfu/ mL) = cell number in each well × virus dilution factor × 10/ volume of added virus fluid (mL).
7
Lentiviral Vector Production and Titration
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Lentiviral vector plasmids and the packaging plasmids (psPAX and pMD.2G) were purchased from Addgene (Watertown). Lentiviral particles with pCDH-EF1-human FGF21, FGF21+GLP1 and GLP1produced through transfection of HEK293T packaging cells with 3rd generation plasmids system. 293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of pMD. 2G, psPAX, and pCDH-EF1 plasmid were 1:2:3. The supernatants were collected at 24 and 48h after transfection, ltered by 0.45 μm lters, and harvested by ultra ltration with 100 kDa spin column (Millipore) at 4°C and 4,000g for 30 min. Lentiviral particles were dispensed in aliquots, and stored at -80 °C until use. Transfection e ciency was examined by EGFP using ow cytometry (Beckman), and viral titer was determined according to the equation: virus titer (pfu/ mL) = cell number in each well × virus dilution factor × 10/ volume of added virus uid (mL).
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