Each immunodepleted plasma sample was concentrated in a 10 kDa Vivaspin 6 concentrator (Sartorius AG, Goettingen, Germany) and reconcentrated in 1 M triethylammonium bicarbonate buffer (TEAB) by centrifugation. The protein concentration in 2 μL of depleted plasma was measured with an infrared-based method (Direct Detect, Merck Millipore, Darmstadt, Germany) according to the protocol provided by the manufacturer. Depleted plasma protein samples were reduced with 30 mM Tris(2-carboxyethyl)phosphine, alkylated with 30 mM iodoacetamide and digested with 0.2 μg/μL trypsin (trypsin:plasma protein ratio of 1:20). To stop the digestion reaction, samples were boiled, then desalted on Strata-X 33 μm polymeric reversed phase columns (Phenomenex) and dried down in a speedvac.
Vivaspin 6 concentrator
Manufactured by Sartorius
Sourced in Germany
The Vivaspin 6 concentrator is a centrifugal device designed for the rapid concentration and desalting of biological samples. It features a vertically oriented membrane to optimize the flow of sample through the device, ensuring efficient sample recovery.
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Lab products found in correlation
Direct detect, by Merck Group (1 mentions)
5 mm bbfo probe, by Bruker (1 mentions)
Ni nta affinity resin, by Qiagen (1 mentions)
Pei max, by Polysciences (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Cary 60 uv visible spectrophotometer, by Agilent Technologies (1 mentions)
Sigma 3 30 ks, by Merck Group (1 mentions)
Vivaspin, by Sartorius (1 mentions)
8 protocols using vivaspin 6 concentrator
1
Depletion and Digestion of Plasma Proteins
2
Nanodisc Characterization by NMR Spectroscopy
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Nanodisc samples were concentrated to ~200 μM in 280 μL in a Vivaspin-6 concentrator with a 30 kDa MWCO (Sartorius). 20 μL D2O was added and gently mixed into the sample. 19F-NMR and 31P-NMR experiments were measured on a Bruker Avance III HD spectrometer operating at 600 MHz 1H nutation frequency using Topspin 3.6.2 and equipped with a Bruker 5-mm BBFO probe. To make direct comparisons with previously published 19F-NMR data50 , 19F-NMR spectra were measured at 280 K. 31P-NMR experiments were measured at 300 K to obtain improved spectral resolution. Temperatures were calibrated from a standard sample of 4% methanol in D4-MeOH.
1-dimensional 19F data were recorded with a data size of 32k complex points, an acquisition period of 360 ms, 16k scans, 120 μs dwell time, and 0.3 s recycle delay for a total experimental time of about 3 hours per experiment. All 31P NMR experiments were acquired with an acquisition time of 900 ms, 2k scans, and 0.3 s recycle delay for a total experiment time of 42 min per experiment.
2-dimensional [19F,19F]-EXSY experiments were recorded with a data size of 120 and 8192 complex points in the indirect and direct dimensions, respectively. We recorded 256 scans for each experiment with 100 ms of mixing time.
1-dimensional 19F data were recorded with a data size of 32k complex points, an acquisition period of 360 ms, 16k scans, 120 μs dwell time, and 0.3 s recycle delay for a total experimental time of about 3 hours per experiment. All 31P NMR experiments were acquired with an acquisition time of 900 ms, 2k scans, and 0.3 s recycle delay for a total experiment time of 42 min per experiment.
2-dimensional [19F,19F]-EXSY experiments were recorded with a data size of 120 and 8192 complex points in the indirect and direct dimensions, respectively. We recorded 256 scans for each experiment with 100 ms of mixing time.
3
Purification of Hexahistidine-tagged Chz1 Protein
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Chz1 was purified via nickel affinity chromatography as follows: a pQE80L plasmid containing Chz1 harboring a N-terminus hexahistidine tag was transformed into E. coli Rosetta 2(DE3)pLysS cells. 3 L of transformed cells were grown in 2xYT media to OD 0.7 and induced with 0.8 mM IPTG overnight at 18 °C. The cells were harvested the next day by centrifugation at 3000 × g for 15 min at 4 °C. The pellets were resuspended in 50 mL wash buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM BME, 1 mM PMSF) with 10 mM imidazole, sonicated, and clarified by centrifugation at 23,000 × g for 20 min at 4 °C. The supernatant incubated with 150 μL Ni-NTA affinity resin (QIAGEN #30210) on the nutator at 4 °C for 3 h. The supernatant was allowed to flow through, and the resin was washed with 5 mL wash buffer containing 10 mM imidazole, 5 mL wash buffer with 40 mM imidazole, and eluted with three rounds of 1.5 mL wash buffer with 200 mM imidazole. The fractions were checked via SDS-PAGE and the cleanest Chz1-containing fraction was concentrated using a 10 kDa cutoff Vivaspin 6 concentrator (Sartorius #VS0601) at 3000 × g at 4 °C. The concentrated Chz1 was dialyzed into storage buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM TCEP), flash frozen in aliquots, and stored at −80 °C. Chz1 concentration was determined to be ~100 μM by UV absorbance at 276 nm.
4
Preparation of Multifunctional Tetrananogels
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Tetrananogels containing Dex‐Mn‐DOTA15 were prepared using a modified method for nanogels containing proteins.17 , 19 The gel material, PEG‐Ac, was synthesized by condensation reactions between tetra‐PEG‐amine and acryloyl chloride. The reacted product was precipitated in diethyl ether on ice, and the suspension was filtered, then dialyzed to purify the PEG‐Ac. The structure of PEG‐Ac was confirmed by 1H NMR.16 A mixture of 200 μL of 100 mg/mL the PEG‐Ac, 50 μL of 400 mM (Mn2+) Dex‐Mn, 50 μL of 16 mg/mL DAB‐Ac, 50 μL of 2 mg/mL of rhodamine, 25 μL of 0.1 mM APS, and 25 μL of 0.1 mM TEMED was stirred for 20 minutes at room temperature. The 3D polymer mesh structure of the gel entraps the Dex‐Mn. After the reaction, the products were recovered using a Vivaspin 6 concentrator with a MWCO of 30 000 (Sartorius, Germany) at 6000 g for 15 minutes at 4°C. Water was added to adjust gels to a final concentration of 100 mM Mn2+ in gel solution.
5
Production and Purification of GPΔTM Proteins
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For production of GPΔTM proteins (wild-type and mutants), Expi293F cells were transfected with pHLsec-GPΔTM using polyethyleneimine (PEI MAX, Polysciences, Warrington, PA) at a mass ratio of 1:3 DNA:PEI MAX. As previously described, a 2:1 ratio of pHLsec-GPΔTM to tagged pHLsec-GPΔTM was transfected to ensure that GPΔTM trimers contained on average a single tagged protomer. The supernatants containing soluble GPΔTM proteins were harvested 5 days post-transfection. The proteins were purified using Ni-NTA agarose beads (Pierce, ThermoFisher Scientific, Waltham, MA). The protein was bound to the column in phosphate-buffered saline (PBS) containing 10mM imidazole, followed by washing with 20mM imidazole in PBS and elution in 200 mM imidazole containing PBS. Following purification, proteins were exchanged to labelling buffer (20 mM HEPES, 50 mM NaCl, pH 7.5) using VivaSpin 6 concentrator (Sartorius AG, Gottingen, Germany).
6
Purification of Histidine-tagged VitR Protein
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The coding region of the vitR gene was PCR-amplified (Table S4 ) and cloned into the pET15b vector (Table S3). The recombinant histidine-tagged protein was overexpressed in E. coli BL21(DE3) by induction with 1 mM isopropyl-D-thiogalactopyranoside (IPTG) for 2 h at 37°C in LB medium. After induction, the soluble fraction containing the His-VitR protein was purified using NTA-resin affinity chromatography in phosphate buffer, according to the manufacturer’s recommendations (Qiagen). After concentration (Vivaspin 6 Concentrator, Sartorius Stedim Biotech) and desalting (PD 10 Desalting Columns, GE Healthcare), the purified VitR protein was resolved by 15% SDS-PAGE.
7
Determination of Entrapment Efficiency and Drug Payload for APO-Loaded Lipid Nanocapsules
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The entrapment efficiency (EE) of APO in APO-LNC formulations was obtained by calculating the free APO concentration in the filtrate using ultrafiltration/centrifugation technique [47] . A 3 ml sample was placed in Vivaspin ® 6 concentrator (MWCO = 100,000, Sartorius, USA) and centrifuged at 6000 rpm for 15 min at 4 °C (Sigma 3-30KS, Sigma Laborzentrifugen GmbH, Germany). The free drug in the filtrate was determined by UVvisible spectrophotometry at 278 nm (Cary 60 UV-visible spectrophotometer, Agilent, Santa Clara, CA, USA). Method validation regarding linearity, limit of detection (LOD), limit of quantitation (LOQ) and % recovery was done. The %EE was calculated from the difference between the initial drug concentration added and the free drug concentration in the filtrate as in Eq. 1: $$EE\%\;=\frac{total\; drug\; amount-unentrapped\; drug }{total\; drug\; amount }*100$$ (1) Measurements were done in triplicate (n = 3).
Drug payload (DL) was then calculated relative to the total dry weight of the LNC formulation (Eq. 2): $$DL=\;\frac{APO\; entrapped\; (mg)}{Total\; dry\; weight\; (g)}$$ (2)
Drug payload (DL) was then calculated relative to the total dry weight of the LNC formulation (Eq. 2): $$DL=\;\frac{APO\; entrapped\; (mg)}{Total\; dry\; weight\; (g)}$$ (2)
8
Purification of Recombinant IgG Antibody
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Prepare the IgG column:
Resuspend the Protein A beads and add 2 mL to a 14 cm chromatography column attached to a retort stand, and allow the column to settle.
Equilibrate the column with 10 mL 1× PBS.
After 5–7 days, collect the culture media and centrifuge at 4500 rpm for 20 min.
Load the supernatant onto the column and collect the flow through for storage on ice.
Wash the column twice with 10 mL 1× PBS.
Elute the antibody from the column with 4.5 mL elution buffer into a 15 mL tube containing 500 μL neutralization buffer.
Concentrate the eluted antibody to <1 mL using a 10 kDa MWCO vivaspin 6 concentrator, according to the manufacturer’s instructions:
Quantify the antibody using spectrophotometry (i.e., Nanodrop), and store the antibody at −20°C for future use.
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