Slide a lyzer g2 dialysis cassette

Manufactured by Thermo Fisher Scientific

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The Slide-A-Lyzer G2 dialysis cassettes are a laboratory equipment designed for the purification and desalting of biological samples. They provide a convenient and efficient method for dialyzing proteins, peptides, and other macromolecules against a desired buffer solution.

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49 protocols using slide a lyzer g2 dialysis cassette

Synthesis and Characterization of Multifunctional Nanoparticles

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PEG derivatives were purchased
from JenKem
Technology. The Pierce Biotin Quantitation Kit and Slide-A-Lyzer G2
Dialysis Cassettes were purchased from Thermo Scientific. All dialyses
employed (10k MWCO, 3 mL capacity) cassettes The mPEG-monophosphates
mPEG750-OPO₃H₂ and mPEG2000-OPO₃H₂ were synthesized according to a literature procedure.^{14 (link)} All the other chemicals were purchased from
Sigma-Aldrich and used without further purification. NaGdF₄ and NaTbF₄ NPs were synthesized following the method
of Li et al.^{35 (link)} with details provided in Supporting Information (SI). The synthesis of
the PAMAM dendrimer with an N-Boc-ethylenediamine core (2 in Scheme 1), followed the strategy described
by Cao et al. with details provided in SI.³⁶

Zhao G., Tong L., Cao P., Nitz M, & Winnik M.A. (2014). Functional PEG–PAMAM-Tetraphosphonate Capped NaLnF4 Nanoparticles and their Colloidal Stability in Phosphate Buffer. Langmuir, 30(23), 6980-6989.

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Synthesis and Characterization of Iron Nanoparticles

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Iron tri(acetylacetonate) (2 mmol), 1,2-tetradecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol), dibenzyl ether, citric acid, diethyl ether, 2-methoxyethylamine, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), thioglycolic acid, hydroxylamine, and phenanthroline were all purchased from Sigma-Aldrich (St. Louis, Missouri). 1,2-dichlorobenzene and N,N’-dimethylformamide were from Acros Organics (Morris Plains, New Jersey). NH₂-PEG (550 Da, 2000 Da, 5000 Da, and 10000 Da) were all obtained from Laysan Bio, Inc. (Arab, Alabama). Slide-A-Lyzer G2 dialysis cassettes, chambered slides, bovine calf serum (BCS) were acquired from Thermo Scientific (Rockford, Illinois). Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffer Saline (DPBS), penicillin, and streptomycin were purchased from Gibco Life Technologies, Inc. (NY, USA).

Park Y.C., Smith J.B., Pham T., Whitaker R.D., Sucato C.A., Hamilton J.A., Bartolak-Suki E, & Wong J.Y. (2014). Effect of PEG Molecular Weight on Stability, T2 contrast, Cytotoxicity, and Cellular Uptake of Superparamagnetic Iron Oxide Nanoparticles (SPIONs). Colloids and surfaces. B, Biointerfaces, 119, 106-114.

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SN-38 Release Kinetics from mAb-Modified NPs

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To determine the release profile of SN-38 from the mAb-modified NPs, samples containing 100 µg of encapsulated SN-38 were resuspended in 3 mL of pH 7.4 PBS (10% HPBCD) and loaded into a 40 kDa cutoff Slide-A-Lyzer G2 dialysis cassettes from Thermo Fisher (Waltham, MA). The cassettes were placed individually in hermetic plastic bags with 10 mL of pH 7.4 PBS (10% HPBCD), at 37 °C, in continuous stirring for 96 h. At sampling times 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 h, buffer from the airtight plastic bags was completely removed and replaced with fresh buffer. Then, 100 µL of the removed buffer was processed as previously described to analyze SN-38 [31 (link)]. The rate of drug release was analyzed by fitting the data into first order equation(1),

ln (100 - Q) = ln 100 - kt

where Q is the percentage of SN-38 released at time t, and k is the release rate constant.

Monterrubio C., Paco S., Olaciregui N.G., Pascual-Pasto G., Vila-Ubach M., Cuadrado-Vilanova M., Ferrandiz M.M., Castillo-Ecija H., Glisoni R., Kuplennik N., Jungbluth A., de Torres C., Lavarino C., Cheung N.K., Mora J., Sosnik A, & Carcaboso A.M. (2017). Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8. Journal of controlled release : official journal of the Controlled Release Society, 255, 108-119.

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Silkworm-Derived Nanomaterial Synthesis

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B. Mori silkworm cocoons were purchased from Wild Fibers, UK. Tetramethyl orthosilicate (98% purity, TMOS), hexadecyltrimethylammonium bromide (98% purity, CTAB), methanol (99.8%, MeOH) and trimethoxysilane (95% purity), 4-pentanoic acid, (≥98% purity), lithium bromide anhydrous, (99.99% purity, LiBr), ammonium hydroxide (28–30%, NH₄OH), sodium carbonate (Na₂CO₃), were procured from Sigma Aldrich. Slide-A-Lyzer™ G2 dialysis cassettes, (3.5 K MWCO, 3–5 mL) were purchased from ThermoFisher. All chemicals were used without further purification. Slide-A-Lyzer™ Dialysis Cassettes with molecular weight cutoffs (MWCOs) of 3.5 kD and volume capacity of 3.5–5 mL was purchased from ThermFisher SCIENTIFIC.

Maleki H, & Huesing N. (2019). Silica-silk fibroin hybrid (bio)aerogels: two-step versus one-step hybridization. Journal of Sol-Gel Science and Technology, 98(2), 430-438.

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Paclitaxel-Loaded PLGA-PEG Nanoparticle Preparation

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PLGA-PEG block copolymers were purchased from Akina, Inc. Paclitaxel was obtained from LC Laboratories (165 New Boston Street, Woburn, MA). DMSO, empty PD-10 columns, calcium chloride, sodium chloride, Slide-A-Lyzer G2 Dialysis Cassettes, Parafilm M™ Laboratory Wrapping Film, and HPLC-purified water were purchased from Thermo Fisher Scientific, Inc. Polyoxyl 20-stearyl ether (Brij 78) was acquired from Uniqema (Wilmington, DE), d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) was purchased from Eastman Chemicals (Kingsport, TN), DSPE-mPEG_2k was acquired from Creative PEGWorks, PLGA (50:50, 33 kDa) was purchased from Lakeshore Biomaterials, and Miglyol 812 was obtained from Sasol (Witten, Germany). HPLC grade acetonitrile was obtained from Acros. All other solvents, Zonyl® FS-300, Pluronic® F-127, and Sepharose CL-4B were purchased from Sigma. Nanoparticle fabrication occurred in a Biotage® Initiator Classic microwave synthesizer. Dynamic light scattering took place in a Zetasizer Nano ZS Particle Analyzer (Malvern Instruments, Inc.). Paclitaxel analysis was performed using High-Performance Liquid Chromatography (Agilent 1260 – Infinity). Particle quantification was performed by thermogravimetric analysis (Discovery TGA, TA Instruments). Transmission Electron Microscopy was carried out using JEOL JEM 1230 TEM.

Dunn S.S., Luft J.C, & Parrott M.C. (2019). Zapped Assembly of Polymeric (ZAP) Nanoparticles for Anti-Cancer Drug Delivery. Nanoscale, 11(4), 1847-1855.

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CYP2B1 and CYP2B6 Enzyme Kinetics

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The primary reaction mixtures contained 250 nM CYP2B1 or CYP2B6, 500 nM CPR, 250 nM cytochrome b₅, 30 ng/μL DLPC, and 5 μM CPS in 50 mM phosphate buffer at pH 7.4. The reactions were initiated by the addition of NADPH to a final concentration of 1 mM or with water for the control reactions. The reaction mixtures were incubated for 1 min before they were placed on ice to stop the reaction. The reactions were then dialyzed against 50 mM potassium phosphate buffer, pH 7.4, in 3 mL Slide-A-Lyzer G2 Dialysis cassettes (Thermo Fisher Scientific). The buffer was exchanged every 15 min over the course of an hour for a total of four buffer exchanges. Following dialysis, a 20 μL aliquot was transferred to a 1 mL secondary reaction solution that contained 0.1 mM 7-EFC and 0.2 mM NADPH in 50 mM potassium phosphate buffer at pH 7.4. The secondary reaction mixture was then incubated for 5 min at 37 °C before it was terminated by the addition of 330 μL of ice-cold acetonitrile. The fluorescence of 7-hydroxy-4-trifluoromethylcoumarin was measured as described above.

D’Agostino J., Zhang H., Kenaan C, & Hollenberg P.F. (2015). Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos. Chemical research in toxicology, 28(7), 1484-1495.

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Formulation of mRNA-loaded LNPs

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5′ methoxyuridine mCherry mRNA (L-7203, TriLink Biotechnologies, San Diego, CA) was formulated into LNPs using the microfluidic setup described in detail elsewhere.^{24 (link)} In brief, stocks of lipids were dissolved in ethanol and mixed in the weight ratio of 20:1 lipid L mRNA to obtain a lipid concentration of 12.5 mM (1.85 mg/mL). The aqueous and ethanol solutions were mixed in a 3:1 volume ratio using a microfluidic apparatus NanoAssemblr (Precision NanoSystems, Vancouver, BC, Canada) at a mixing rate of 12 mL/min. LNPs were dialyzed overnight in PBS using Slide-A-Lyzer G2 dialysis cassettes (Thermo Fisher) with a molecular weight cutoff of 10 kDa. The size of the LNPs was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), and the concentration and encapsulation of mRNA were determined using the RiboGreen assay. The final ratio of lipids by molar percentage was 50:38.5:10:1.5 (MC3:DSPC:PEG-DMG 2000) with a lipid to RNA mass ratio of 20:1.

Ross-Thriepland D., Bornot A., Butler L., Desai A., Jaiswal H., Peel S., Hunter M.R., Odunze U., Isherwood B, & Gianni D. (2020). Arrayed CRISPR Screening Identifies Novel Targets That Enhance the Productive Delivery of mRNA by MC3-Based Lipid Nanoparticles. Slas Discovery, 25(6), 605-617.

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Cloning and Purification of SRP9/14 Protein

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SRP9/14 expression plasmid was created by amplification of a coding segment of p14-9VN (Addgene plasmid # 50930) using primers SRP14F NcoI and SRP9R XhoI (Supplementary Table S1). The amplicon was inserted into NcoI/XhoI sites of pET-28a (Novagen). The hybrid protein was expressed in BL21 (DE3) pLysS Competent Cells (Promega). The cells were grown to an OD of 0.8 and protein expression was induced by 1 mM IPTG at 37°C for 3 h. Bacterial pellets were dissolved in buffer A (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10% glycerol (v/v) and 3.6 mM β-mercaptoethanol) containing the cOmplete™, an EDTA-free Protease Inhibitor Cocktail (Roche) and were homogenized using SONOPULS GM Mini 20 (Bandelin Electronic). Proteins were purified using the Ni Sepharose 6 Fast Flow beads (GE Healthcare), washed five times with buffer A containing 20 mM imidazole and eluted with buffer A with 300 mM imidazole. The purified heterodimer was dialyzed against a storage buffer (10 mM Tris–HCl, pH 8.0, 140 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10% glycerol (v/v) and 1 mM β-mercaptoethanol) using the Slide-A-Lyzer™ G2 Dialysis Cassettes (ThermoFisher) at 4°C overnight and stored at –80°C.

Borovská I., Vořechovský I, & Královičová J. (2023). Alu RNA fold links splicing with signal recognition particle proteins. Nucleic Acids Research, 51(15), 8199-8216.

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Silk-based Composite Material Synthesis

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B. mori silkworm cocoons were purchased from Wild Fibres, UK. Methyl trimethoxysilane (98% purity, MTMS), hexadecyltrimethylammonium bromide (98% purity, CTAB), methanol (99.8%, MeOH), trimethoxysilane (95% purity), 4-pentenoic acid, (≥98% purity), anhydrous lithium bromide (99.99% purity, LiBr), ammonium hydroxide (28–30%, NH₄OH), sodium carbonate (Na₂CO₃), methylene blue (dye content > 82%, MB) were obtained from Sigma Aldrich. Acetone, dimethylformamide (DMF), toluene, pump oil were purchased from VWR International. Slide-A-Lyzer™ G2 dialysis cassettes, (3.5 K MWCO, 3–5 mL) were purchased from Thermo Fisher Scientific Inc. All chemicals were used without further purification.

Maleki H., Whitmore L, & Hüsing N. (2018). Novel multifunctional polymethylsilsesquioxane–silk fibroin aerogel hybrids for environmental and thermal insulation applications †Electronic supplementary information (ESI) available: Additional data belong to [Si]17.5, SEM micrographs, FT-IR, N2 adsorption–desorption isotherms, TGA-DTA analysis and so on. See DOI: 10.1039/c8ta02821d. Journal of Materials Chemistry. a, 6(26), 12598-12612.

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Refolding and Solubilization of IL-18 Proteins

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The sonicated cell pellet was washed with 25 ml PBS thrice and centrifuged at 10,000 rpm for 10 min. The pellet was then collected, weighed, and resuspended in solubilization buffer (base buffer (50 mM Tris-HCl, 150 mM sodium chloride (NaCl), 100 mM glycine, 20% glycerol, pH 8.0) containing 8 M urea) at a final concentration of 2–8 mg/ml. The suspension was mixed in an orbital shaker for 30 min, and after centrifugation at 15,000 rpm for 15 min, the supernatant was filtered through a 0.45 µm syringe filter. The supernatant was dialyzed using slide-A-Lyzer G2 dialysis cassettes (Thermo Scientific, Waltham, MA, USA) in 1 L dialysis buffer (base buffer containing 6 M urea) for 4 h at 4 °C. During this step, 500 mL of base buffer was added to the dialysis buffer every 6 or 12 h 4 times. Finally, the dialysis cassette with the solubilized IL-18 inclusion bodies was placed in 2 ml base buffer for 6 h. The refolded IL-18 proteins (WT and mutants) were harvested and stored at 4 °C until further use.

Saetang J., Roongsawang N., Sangkhathat S., Voravuthikunchai S.P., Sangkaew N., Prompat N., Srichana T, & Tipmanee V. (2022). Surface cysteine to serine substitutions in IL-18 reduce aggregation and enhance activity. PeerJ, 10, e13626.

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