Ultrafiltration

Manufactured by Merck Group

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Ultrafiltration is a membrane-based separation process that utilizes pressure to separate molecules or particles from a solution based on their size or molecular weight. It is commonly used for the purification, concentration, or fractionation of macromolecules such as proteins, peptides, or polysaccharides.

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42 protocols using ultrafiltration

Purification of Recombinant Protein from E. coli

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Plasmids confirmed to have the correct sequence were electroporated into E. coli BL21 (DE3) (Novagen, Madison, WI, USA) competent cells. The cells were incubated in 1 mL LB for 1 h, spread on LB plates with carbenicillin (50 μg/mL) and incubated overnight. Single colonies were picked and grown overnight in 3 mL LB with carbenicillin (50 μg/mL). The antibiotic-selected culture was inoculated into 500 mL LB containing 50 μg/mL carbenicillin. The expression culture was induced with IPTG (0.4 mM) until OD600 reached approximately 0.4–0.6, and further incubated (30 °C, 3 h). Bacterial cells were harvested by centrifugation (10,000 rpm, 4 °C, 20 min). The pelleted bacteria were suspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication. The soluble protein was applied to Ni-NTA resin (QIAGEN) and eluted by affinity tag cleavage following incubation with enterokinase (5 μL, 2.0 μg/mL, 4 °C, 36 h). The protein was concentrated via ultrafiltration (Millipore, Burlington, MA, USA) and further purified by size exclusion chromatography. The final purity of the protein was determined by SDS-PAGE.

Zhang Y., Zhou J., Ardejani M.S., Li X., Wang F, & Orner B.P. (2017). Designability of Aromatic Interaction Networks at E. coli Bacterioferritin B-Type Channels. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(12), 2184.

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Synthesis of Fluorescent Gold Nanoclusters

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An amount of 100 mL freshly dissolved GSH (30 mM) was mixed with an equal volume of HAuCl₄·3H₂O solution (20 mM) under stirring (500 rpm) at room temperature for 10 min. The mixture was heated to 70 °C for 12 h in a water bath with gentle stirring (500 rpm). Then, the reaction solution was kept at room temperature and shielded from light for another 12 h. After that, the generated orange-emitting AuNCs were purified according to our previous report [21 (link)]. The last step is to further purify the AuNCs aqueous solution using ultrafiltration (Millipore, Burlington, MA, USA, MWCO: 3 kDa) to remove the unreacted free ions and stock at 4 °C.

Meng C., Liu Y., Ming Y., Lu C., Li Y., Zhang Y., Su D., Gao X, & Yuan Q. (2024). Enhancing Liver Delivery of Gold Nanoclusters via Human Serum Albumin Encapsulation for Autoimmune Hepatitis Alleviation. Pharmaceutics, 16(1), 110.

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Purification of BP-Specific IgG Subclasses

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Serum samples were collected from three patients with active BP (BP1, BP2, and BP3). NC16A-specific total IgG were purified from BP patient sera using a protein G column, followed by an NC16A-specific glutathione sepharose column as described [30 (link)]. NC16A-specific IgG1,3 and 4 were purified from NC16A-specific total IgG using human IgG4-specific columns (Amersham Biosciences). The concentration and purity of NC16A-specific IgG4 (eluate) and NC16A-specific IgG1 and 3 (flow through) were determined by human IgG subclass-specific ELISA (Southern Biotechology). The purity of NC16A-specific IgG1,3 and IgG4 were 95% and 98%, respectively. Purified IgG and IgG subclass fractions were concentrated by ultrafiltration (Millipore) and used for in vitro and in vivo experiments.

Zuo Y., Evangelista F., Culton D., Guilabert A., Lin L., Li N., Diaz L, & Liu Z. (2016). IgG4 autoantibodies are inhibitory in the autoimmune disease bullous pemphigoid. Journal of autoimmunity, 73, 111-119.

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Fluorescent Labeling of Spy-Tagged Proteins

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Example 9

Purified His₆-DDB1ΔB-His₆-3C-Spy-CRBN or His₆-Spy-BRD4_BD1was incubated overnight at 4° C. with BODIPY-FL labelled SpyCatchers_50Cprotein at stoichiometric ratio. Protein was concentrated and loaded on the ENrich SEC 650 10/300 (Bio-rad) size exclusion column and the fluorescence monitored with absorption at 280 and 490 nm. Protein peak corresponding to the labeled protein was pooled, concentrated by ultrafiltration (Millipore), flash frozen (˜9.6 μM for His₆-DDB1ΔB-His₆-3C-Spy-CRBN_{BODIPY SpyCatcher}or ˜22 uM for His₆-Spy-BRD4_BD1) in liquid nitrogen and stored at −80° C.

US11505560B2. Heterobifunctional compounds with improved specificity (2022-11-22). DANA-FARBER CANCER INSTITUTE, INC. [US]. Inventors: Radoslaw P. Nowak [US], Eric S. Fischer [US], Nathanael S. Gray [US], Tinghu Zhang [US], Zhixiang He [US], Brian Groendyke [US].

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CRISPR Array Verification by PCR Sequencing

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To confirm the genomic differences observed between CRISPR arrays of the two African isolates based on Illumina sequence data, three primer pairs were designed for PCR amplification and sequencing of the CRISPR locus in three overlapping fragments: (i) L1F-CGTAAACGTCTGTTAGATGATGG and sp15R-AAACCATTCTACGGAGAAC; (ii) sp17F-GATGTAATAAGAGTTGTTGCG and sp5R-TCGGATTTATGAGGTGATCCC; and (iii) sp7F-CATAGATCACACATACAGGGC and L1R-TGAGCGCCCATGTTGTCTCCG. PCR conditions for all amplification reactions were as follows: initial denaturation at 94 °C for 5 min; 30 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30s; and final extension at 72 °C for 5 min. PCR products were purified by ultrafiltration (Millipore), and nucleotide sequences were obtained using the PCR primers and BigDye Terminator v1.1 chemistry (Applied Biosystems, Foster City, CA) on an ABI 3730XL apparatus (Applied Biosystems, Foster City, CA). Sequence traces were edited and assembled using BioNumerics.

Breurec S., Criscuolo A., Diancourt L., Rendueles O., Vandenbogaert M., Passet V., Caro V., Rocha E.P., Touchon M, & Brisse S. (2016). Genomic epidemiology and global diversity of the emerging bacterial pathogen Elizabethkingia anophelis. Scientific Reports, 6, 30379.

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Recombinant Expression and Purification of DDB1ΔB and CRBN

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Human DDB1ΔB and human CRBN were cloned in pAC-derived vectors^{30 (link)} and recombinant proteins were expressed as N-terminal His₆ (DDB1ΔB) or StrepII-Avi (CRBN) fusions in Trichoplusia ni High-Five insect cells using the baculovirus expression system (Invitrogen). For purification, cells were resuspended in buffer containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0, 200 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1x protease inhibitor cocktail (2 μg/ml Aprotinin, 10μM Bestatin, 2μM E-64, 10μM Leupeptin, 1μM Pepstatin A and 10μM 1,10 - Phenanthrolin and 1μM Phosphoramidon) and lysed by sonication. Following ultracentrifugation, the soluble fraction was passed over Strep-Tactin XT (IBA, 2-4030-025) resin and eluted with wash buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP) supplemented with 50 mM biotin (MedChemExpress, HY-B0511). The affinity-purified protein was subjected to ion exchange chromatography (Poros 50HQ) followed by size exclusion chromatography (16/60 S200, GE) in 50 mM HEPES pH 7.4, 200 mM NaCl and 1 mM TCEP. Biotinylation of was performed as previously described.^{31 (link)} The protein-containing fractions were concentrated using ultrafiltration (Millipore) and flash frozen in liquid nitrogen at 40-120 μM concentration and stored at −80°C.

Powell C.E., Du G., Che J., He Z., Donovan K.A., Yue H., Wang E.S., Nowak R.P., Zhang T., Fischer E.S, & Gray N.S. (2020). Selective Degradation of GSPT1 by Cereblon Modulators Identified via a Focused Combinatorial Library. ACS chemical biology, 15(10), 2722-2730.

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Isolation and Characterization of Exosomes from Bone Marrow-Derived Mesenchymal Stem Cells

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The BMMSCs were cultured in DMEM with exosome-free FBS (Hyclone, South Logan, UT, USA). The culture medium was collected, filtered using a 0.22-μM cell filter, and then concentrated using ultrafiltration (Millipore Corp., Bedford, MA, USA). Exosomes were obtained following the manufacturer’s protocol of exosome isolation kit (Invitrogen, Carlsbad, CA, USA). A total of 10 μL extracted exosomes were diluted with equal volume of PBS and then negatively stained using 3% sodium phosphotungstate solution for 1 min. After washing with distilled deionized water and drying at room temperature, the exosomes were observed and photographed under a transmission electron microscope (Hitachi, Tokyo, Japan).
Dynamic light scattering was performed to measure exosome particle diameter. Particle size distribution was analyzed using Zetasizer Nano ZS90 (Malvern Panalytical, UK). The diluted samples in PBS in the ratio of 1:20 were manually loaded into the sample chamber. Three videos (60 s) were recorded of each sample. Data was analyzed using DTS v5.10 software (Malvern Panalytical, UK).

Li R., Zhao K., Ruan Q., Meng C, & Yin F. (2020). Bone marrow mesenchymal stem cell-derived exosomal microRNA-124-3p attenuates neurological damage in spinal cord ischemia-reperfusion injury by downregulating Ern1 and promoting M2 macrophage polarization. Arthritis Research & Therapy, 22, 75.

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Fluorescent Spycatcher Labeling Protocol

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Purified Spycatcher_S50C protein was incubated with DTT (8 mM) at 4°C for 1 h. DTT was removed using a ENRich SEC650 10/300 (Bio-rad) size exclusion column in a buffer containing 50 mM Tris pH 7.5 and 150 mM NaCl, 0.1mM TCEP. BODIPY-FL-maleimide (Thermo Fisher) was dissolved in 100% DMSO and mixed with Spycatcher_S50C to achieve 2.5 molar excess of BODIPY-FL-maleimide. SpyCatcher_S50C labelling was carried out at room temperature (RT) for 3 h and stored overnight at 4°C. Labelled Spycatcher_S50C was purified on a ENRich SEC650 10/300 (Bio-rad) size exclusion column in 50 mM Tris pH 7.5, 150 mM NaCl, 0.25 mM TCEP and 10% (v/v) glycerol, concentrated by ultrafiltration (Millipore), flash frozen (~40 μM) in liquid nitrogen and stored at -80°C.

Nowak R.P., DeAngelo S.L., Buckley D., He Z., Donovan K.A., An J., Safaee N., Jedrychowski M.P., Ponthier C.M., Ishoey M., Zhang T., Mancias J.D., Gray N.S., Bradner J.E, & Fischer E.S. (2018). Plasticity in binding confers selectivity in ligand induced protein degradation. Nature chemical biology, 14(7), 706-714.

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Recombinant Human Glucokinase Expression

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The full-length human glucokinase (GenBank: BC001890.1) was cloned into the pET-26b vector (Novagen). The recombinant plasmid was transformed into E. Coli strain BL21(DE3) (Invitrogen) after verified by sequencing. The recombinant GK proteins were over-expressed and induced by 0.4 mM Isopropyl-β-D-Thiogalactopyranoside (Beijing Dingguo Changsheng Biotechnology Co. Ltd., Beijing, China) at 20 °C for 16 h. Cells were harvested and lysed by ultrasonification on ice in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, and 5 mM β-mercaptoethanol. Soluble C-terminally hexa-histidine tagged GK was purified by a Ni²⁺-chelating column (Qiagen) following by a size exclusion chromatography on a Superdex 300GL column with a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl. The target protein was finally concentrated to the appropriate concentration by ultra filtration (Millipore).

Min Q., Cai X., Sun W., gao F., Li Z., Zhang Q., Wan L.S., Li H, & Chen J. (2017). Identification of mangiferin as a potential Glucokinase activator by structure-based virtual ligand screening. Scientific Reports, 7, 44681.

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Arabidopsis Seedling Growth Assay for Auxin-like Activity

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The potential auxin-like activity of the compounds was evaluated by the Arabidopsis seedling growth test as previously described [17 (link),18 (link)]. Seeds of Arabidopsis Col-0 were surface sterilized with bleach solution (5% (w/v) Ca(OCl)₂ + 0.02% (v/v) Triton X-100 in 80% EtOH) for 10 min by continuous shaking, washed 2 times with 80% EtOH, then with 100% EtOH and finally 3 times with sterile distilled water. Square Petri dishes (12 × 12 cm) with 15 seeds and 80 mL modified MS agar medium [41 (link)] supplemented with three different concentrations (0.1 mM, 0.5 mM and 1 mM) of tested compounds (HEA-pABA and HEA-pNBA, respectively) as well as IAA as a reference control were used. All tested compounds were dissolved in 5 mM MES (2-[N′-morpholino] ethanesulfonic acid) buffer (pH 6), sterilized by ultrafiltration (Millipore) and added to MS agar control (∼50 °C). Five repetitions for control and for each tested compound were performed. For breaking dormancy and synchronizing germination, Petri dishes were horizontally stratified for 2 days at 4 °C in the dark and then transferred to the growth chamber at 22 °C and grown in vertical position under 16 h light/8 h dark photoperiod (220 µmol m⁻²s⁻¹ photosynthetic photon flux density) for 10 days.

Sumalan R.L., Halip L., Maffei M.E., Croitor L., Siminel A.V., Radulov I., Sumalan R.M, & Crisan M.E. (2021). Bioprospecting Fluorescent Plant Growth Regulators from Arabidopsis to Vegetable Crops. International Journal of Molecular Sciences, 22(6), 2797.

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