Polypropylene column

Manufactured by Qiagen

Sourced in Germany

Polypropylene columns are laboratory equipment designed for various separation and purification processes. They are made of polypropylene, a durable and inert plastic material. The columns provide a contained environment for the efficient handling and manipulation of liquids and samples during experimental procedures.

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Lab products found in correlation

Ni nta agarose, by Qiagen (2 mentions) Amicon ultra 30 k centrifugal filter, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Complete protease inhibitor cocktail tablet, by Roche (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Nickel nitrilotriacetic acid agarose, by Qiagen (1 mentions) Glutathione sepharose 4 fast flow, by Cytiva (1 mentions) Fastgene rna premium kit, by Nippon Genetics (1 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions) Expi293f cells, by Thermo Fisher Scientific (1 mentions) Imidazole, by Merck Group (1 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions) Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions) Protein assay, by Bio-Rad (1 mentions) Nickel nitrilotriacetic acid ni nta agarose, by Qiagen (1 mentions) Oligonucleotide, by Integrated DNA Technologies (1 mentions) Ultra centrifugal filters, by Merck Group (1 mentions) Vent dna polymerase and restriction enzymes, by New England Biolabs (1 mentions) Zymolase 20t, by MP Biomedicals (1 mentions)

12 protocols using polypropylene column

Purification of Plant-Produced Antibody

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About 100 g of infiltrated leaves were harvested 4 days after agroinfiltration and the leaves were homogenized with 200 ml PBS buffer. The plant crude extract was centrifuged at 26,000 × g at 4°C for 40 min and clarified with a 0.45-μm membrane filter. The resulting supernatant was purified by protein A affinity resin (Expedeon, United Kingdom) packed in a polypropylene column (Qiagen, Germany) with 15 mm column diameter. The proteins were washed with PBS buffer and the recombinant antibody was eluted using 0.1 M glycine at pH 2.7 and neutralized with 1.5 M Tris-HCl pH 8.8 to final pH 7.4. Purified plant-produced antibody was buffer exchanged and concentrated using Amicon^® Ultra (30 K) centrifugal filter (Merck, Germany) according to the instructions from the manufacturer. Purified plant-produced antibody was quantified by ELISA and used for further experiments.

Phakham T., Bulaon C.J., Khorattanakulchai N., Shanmugaraj B., Buranapraditkun S., Boonkrai C., Sooksai S., Hirankarn N., Abe Y., Strasser R., Rattanapisit K, & Phoolcharoen W. (2021). Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System. Frontiers in Plant Science, 12, 736299.

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Recombinant Protein Purification from E. coli

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Recombinant protein expression was induced in

E. coli

BL21 cultures at OD₆₀₀ 0.5–0.7 by adding IPTG at a final concentration of 1 mM, and continuing incubation for 4 hr at 37°C with shaking at 220 rpm. Cells were harvested by centrifugation (5000 × g, 10 min), and the pellet was lysed in 2 ml Buffer B (8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris–HCl, pH 7.0) supplemented with 0.1 M phenylmethanesulfonylfluoride (PMSF) at room temperature for 4 hr. Cell debris was removed by centrifugation (12,000 × g, 15 min). Ni‐NTA agarose (Qiagen; 300 μl) was added to 2 ml lysis supernatant, mixed gently at room temperature for 25 min, and then transferred to a polypropylene column (Qiagen). The column was washed with 4 ml Buffer B (supplemented with 20 mM imidazole) and then twice with Buffer C (1 M NaCl, 10% EtOH, 2% Tween‐20) supplemented with 20 mM imidazole. Protein was eluted six times with Buffer D (Buffer B, supplemented with 100 mM imidazole). All of the washes and eluted fractions were collected and analysed by SDS‐PAGE. Fractions containing recombinant proteins were pooled and dialysed against dH₂O supplemented with protease inhibitor cocktail (Sigma). Dialysed protein solutions were freeze‐dried, suspended in dH₂O, and the protein concentrations determined using the Bradford assay (BioRad Laboratories, Hemel Hempstead, Herts, UK).

Haworth J.A., Jenkinson H.F., Petersen H.J., Back C.R., Brittan J.L., Kerrigan S.W, & Nobbs A.H. (2016). Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix. Cellular Microbiology, 19(1), e12667.

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Glycated Peptide Enrichment by BAC

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BAC was performed as described by Frolov and Hoffmann (40 (link)) with changes. m-Aminophenylboronic acid-agarose (1 ml) was resuspended in aq. 250 mm NH₄CH₃COO, 50 mm Mg(CH₃COO)₂ × 6 H₂O, pH 8.1, and packed in a 1-ml polypropylene column (85 × 7.5 mm; Qiagen GmbH, Hilden, Germany). 200 μg of the lyophilized tryptic digest was reconstituted in 20% (v/v) aq. CH₃CN and diluted with ice-cold washing buffer to a final volume of 500 μl. After washing out the unbound fraction with 13.5 ml of loading buffer, glycated peptides were eluted in two steps with aq. CH₃COOH (0.1 m, 8 ml; then 0.2 m, 2 ml) at 37 °C. Eluates were lyophilized and stored at −20 °C until further analysis.

Bilova T., Lukasheva E., Brauch D., Greifenhagen U., Paudel G., Tarakhovskaya E., Frolova N., Mittasch J., Balcke G.U., Tissier A., Osmolovskaya N., Vogt T., Wessjohann L.A., Birkemeyer C., Milkowski C, & Frolov A. (2016). A Snapshot of the Plant Glycated Proteome: STRUCTURAL, FUNCTIONAL, AND MECHANISTIC ASPECTS. The Journal of Biological Chemistry, 291(14), 7621-7636.

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Affinity Purification of SIV NS1 Protein

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SRECs’ were infected with SIV/SK-544 at an MOI of 2. At 16 h post infection (h.p.i.), cells were harvested in cell lysis buffer (Cell Signaling Technology) with protease inhibitor (Complete protease inhibitor cocktail tablets – Roche Diagnostics Corporation). The lysate was sonicated and then clarified by centrifugation at 16200 × g for 10 min at 4°C. The Strep-tactin sepharose resin (IBA) was washed three times with four column volume (CV) of cell lysis buffer and then was added to the clarified lysate and incubated at 4°C overnight. Next, the lysate-sepharose mixture was added to a polypropylene column (Qiagen) and the sepharose was washed extensively with wash buffer [100 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA]. The Strep-tag NS1 protein complex was then eluted from the sepharose resin with 3 CV of elution buffer (wash buffer with 2.5 mM desthibiotin). Protease inhibitor cocktail was added to the eluent to prevent degradation of the proteins and the proteins present in the complex were identified by LC-MS/MS at the University of Victoria-Genome BC Proteomics Centre.

Thulasi Raman S.N, & Zhou Y. (2016). Networks of Host Factors that Interact with NS1 Protein of Influenza A Virus. Frontiers in Microbiology, 7, 654.

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Recombinant Headless HA Stalk Purification

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Plasmids encoding the recombinant headless HA stalk were provided by Adrian McDermott and Barney Graham from the Vaccine Research Center at the National Institutes of Health. The headless HA stalk protein was expressed in 293F cells and purified using nickel-nitrilotriacetic acid agarose (no. 1018244; Qiagen) in 5-ml polypropylene columns (no. 34964; Qiagen), washed with pH 8 buffer containing 50 mM Na₂HCO₃, 300 mM NaCl, and 20 mM imidazole, and then eluted using pH 8 buffer containing 50 mM Na₂HCO₃, 300 mM NaCl, and 300 mM imidazole. Purified protein was buffer exchanged into phosphate-buffered saline (PBS; no. 21-031-CM; Corning). Following purification, the headless HA stalk proteins were biotinylated using the Avidity BirA-500 kit (no. BirA500) and stored in aliquots at −80°C. Plasmids encoding the recombinant chimeric (c6/H1) HA were provided by Florian Krammer (Mt. Sinai). The detailed protocol for expression of this protein is published elsewhere (46 (link)). In brief, the c6/H1 HA protein was expressed in High Five baculovirus cells and purified using the same methods referenced for the headless HA stalk protein. Purified protein was buffer exchanged into PBS (no. 21-031-CM; Corning) and stored in aliquots at −80°C.

Christensen S.R., Toulmin S.A., Griesman T., Lamerato L.E., Petrie J.G., Martin E.T., Monto A.S, & Hensley S.E. (2019). Assessing the Protective Potential of H1N1 Influenza Virus Hemagglutinin Head and Stalk Antibodies in Humans. Journal of Virology, 93(8), e02134-18.

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Recombinant ADD Domain Purification

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Complementary DNAs (cDNAs) were generated using 1 μg total RNA from the ovaries as templates using FastGene RNA Premium kit (NIPPON Genetics). cDNA fragments encoding either wild-type or mutated ADD domain (residues 472–605) were amplified by PCR and cloned into pGEX6p-1 vector. The primers used for cloning are listed in S9 Table. The glutathione S-transferase fusion proteins were expressed in Escherichia coli strain Rosetta. The cultures with optical density of 0.6–0.8 were added with 0.2 mM isopropyl-β-D-thiogalactopyranoside and incubated at 20°C for 15 hours. After sonication, the supernatant of cell lysate was mixed with Glutathione Sepharose 4 Fast Flow (Cytiva) and applied onto columns for gravity-flow chromatography (Polypropylene Columns, QIAGEN). Then, the fusion proteins were recovered with elution buffer (50 mM Tris-HCl, 10 mM glutathione, pH 8.0). The eluted proteins were purified by Amicon Ultra Centrifugal Filters and subjected to SDS-PAGE followed by Coomassie blue staining for product size confirmation.

Uehara R., Au Yeung W.K., Toriyama K., Ohishi H., Kubo N., Toh H., Suetake I., Shirane K, & Sasaki H. (2023). The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes. PLOS Genetics, 19(8), e1010855.

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Purification of SARS-CoV-2 Proteins from HEK Cells

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Purified plasmids were used to transfect high density (4–5 × 10⁶ viable cells/mL) cultures of suspension-adapted human embryonic kidney (HEK) cells (Expi293F™ cells, Thermo Scientific Inc., kindly donated by Dr. Jesus Hernandez at the Immunology Laboratory of the Research Center for Food and Development, Mexico) using the ExpiFectamine™ 293 Transfection Kit (Gibco, A14524) and the Expi293 Expression Medium supplemented with GlutaMAX™ following the manufacturer’s instructions. Cell-free supernatants containing soluble SARS-CoV-2 proteins were harvested and concentrated using Amicon^® Ultra-15 centrifugal filters with a 100 kDa cut-off for the full-length S [∼190 kDa molecular weight (mw)] and the N (∼114 mw), or the 10 kDa cut-off filters for the RBD (∼30 kDa mw) and purified following standard protocols for His-tagged protein purification using Ni-NTA agarose (QIAGEN) packed on polypropylene columns (QIAGEN), and imidazole (Sigma) for washing and elution buffers (21 (link)). Eluted proteins were buffer exchanged into sterile PBS using either Amicon Filters with 10 kDa mw cut-off for the RBD or 100 kDa for S or N and quantified using a standard Biuret Protein Assay with BSA as standard protein, then stored at −80°C until further use.

Puerta-Guardo H., Parra-Cardeña M., Peña-Miranda F., Flores-Quintal F., Granja-Pérez P., Villanueva-Jorge S., González-Losa R., Conde-Ferraez L., Gómez-Carballo J., Vazquez-Prokopec G., Earnest J.T., Manrique-Saide P, & Ayora-Talavera G. (2022). Human IgG antibody responses to severe acute respiratory syndrome coronavirus 2 viral antigens receptor-binding domain, spike, and nucleocapsid, in vaccinated adults from Merida, Mexico. Frontiers in Medicine, 9, 916241.

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Recombinant GH28-1 Purification from Pichia

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GH28-1 was expressed in the yeast Pichia pastoris as previously described (76 (link)). The His₆-tagged enzyme was purified using HisPur Cobalt Resin (Thermo Scientific) following the manufacturer’s instructions for the “Batch Method” with slight modifications. The enzyme-containing supernatant from the yeast expression was diluted with an equal volume of Equilibration/Wash Buffer and incubated with the cobalt resin for ∼2–6 h under constant stirring at 4 °C. At room temperature, the mixture was passed over 5 mL Polypropylene Columns (Qiagen), which retained the resin including captured proteins. After washing with 20 column volumes of Equilibration/Wash Buffer, a multistep elution was performed. Two column volumes of elution buffer were incubated on the column for 5 min, drained by gravity flow, and then reapplied to the column. This was repeated three times per elution for four elution fractions. All elution fractions were pooled and a buffer exchange into LiChrosolv ultrapure water (Merck) was carried out using Amicon Ultra-15 Centrifugal Filter Units (Merck) (10 kDa MWCO). The protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad) with the help of a bovine serum albumin (BSA) standard curve. The protein solutions were frozen in individual aliquots in liquid nitrogen and stored at −20 °C until further use.

Kirsch R., Okamura Y., Haeger W., Vogel H., Kunert G, & Pauchet Y. (2022). Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival. Proceedings of the National Academy of Sciences of the United States of America, 119(40), e2205857119.

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Recombinant Protein Production Protocol

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All the chemicals were purchased from commercial sources and were either analytical grade or molecular grade. The growth media components were obtained from BD Difco (Franklin Lakes, NJ, USA) and Himedia (Mumbai, India). The amino acids, glutathione (GSH) (reduced), β‐nicotinamide adenine dinucleotide phosphate sodium salt hydrate (NADP), glucose 6‐phosphate (G6P), mevalonolactone and 6‐phosphogluconic dehydrogenase from yeast were obtained from Merck (Darmstadt, Germany). Zymolase‐20T was obtained from MP biomedicals (USA). Ultra centrifugal filters were obtained from Merck (Burlington, MA, USA). Oligonucleotides were obtained from Integrated DNA Technologies (IDT) and Merck (Bangalore, India). Vent DNA polymerase and restriction enzymes were obtained from New England Biolabs (Ipswich, MA, USA). Plasmid miniprep and gel/PCR clean‐up kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The NADPH kit was obtained from Promega (Madison, WI, USA), nickel‐nitrilotriacetic acid agarose (Ni‐NTA), and polypropylene columns were obtained from Qiagen (Hilden, Germany).

Adusumilli S.H., Alikkam Veetil A., Choudhury C., Chattopadhyaya B., Behera D, & Bachhawat A.K. (2024). Glucose 6‐phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production. FEBS Open Bio, 14(3), 410-425.

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Recombinant Protein Production in HEK293-6E

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All expression plasmids were sequence verified and recombinant proteins produced by transiently transfecting HEK293-6E cells. Plasmids encoding bait proteins were co-transfected with a plasmid encoding secreted BirA in a 9:1 ratio as described [56 ]. HEK293-6E cells were maintained in Freestyle medium (Invitrogen) supplemented with 25 μg/mL G418 (Invitrogen) and 0.1% Kolliphor. Transfections were left for 5 days, and supernatants were harvested and filtered through a 0.2-μm filter. Supernatants containing prey proteins were used neat or diluted without purification while those containing bait proteins were subjected to His-tag affinity purification. Supernatants containing biotinylated bait proteins were incubated with Ni-NTA agarose beads (Jena Bioscience) overnight at 4 °C with constant rotation. One hundred microliters of beads was used for every 50 mL of supernatant. Polypropylene columns (Qiagen) were equilibrated with 2 mL binding buffer (20 mM sodium phosphate buffer, 0.5 mM NaCl, 40 mM imidazole) before addition of the bead-supernatant mixture. Beads were washed with 5 mL binding buffer and proteins eluted in 500 μL of elution buffer (20 mM sodium phosphate buffer, 0.5 mM NaCl, 400 mM imidazole) by incubating for 30 min at room temperature.

Chong Z.S., Ohnishi S., Yusa K, & Wright G.J. (2018). Pooled extracellular receptor-ligand interaction screening using CRISPR activation. Genome Biology, 19, 205.

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