Poly prep column

Manufactured by Bio-Rad

Sourced in United States

The Poly-Prep column is a small-scale gravity-flow chromatography column designed for protein purification. It features a polyethylene body and a porous polyethylene bed support. The column has a 5 mL bed volume capacity and is suitable for a variety of resins and buffers used in affinity, ion exchange, or size exclusion chromatography.

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Lab products found in correlation

Ni sepharose 6 fast flow resin, by GE Healthcare (2 mentions) Sonicator, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Arcticexpress de3, by Agilent Technologies (2 mentions) Bl21 codonplus de3 ripl, by Agilent Technologies (2 mentions) 400 nm diameter polycarbonate filter, by Avestin (2 mentions) Heparin sepharose, by Abcam (2 mentions) Protein a sepharose beads, by Merck Group (1 mentions) Diethylaminoethyl deae sephacel, by GE Healthcare (1 mentions) Superose 6 increase 10 300 column, by GE Healthcare (1 mentions) Amphipol a8 35, by Anatrace (1 mentions) Amicon ultra 0.5 ml 100 k, by Merck Group (1 mentions) Bio beads sm 2, by Bio-Rad (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Protease and phosphatase inhibitor, by Cell Signaling Technology (1 mentions) 6 well culture dishes, by Greiner (1 mentions) Pdisplay bira er, by Addgene (1 mentions) Superdex 200 10 300 column, by GE Healthcare (1 mentions) Ni2 sepharose resin, by GE Healthcare (1 mentions) Protein a sepharose 4 fast flow beads, by GE Healthcare (1 mentions)

Close spelling variants of this product

Poly-Prep chromatography columns (108 citations) Poly-Prep (9 citations) BS Poly-prep columns (7 citations) Poly-Prep Prefilled Chromatography Columns (2 citations)

27 protocols using poly prep column

Maleimide-Activated Sepharose 4B Functionalization

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Sepharose 4B: Maleimide-activated Sepharose 4B resin (2 mL, 50% vol. slurry in water, 0.025 mmol) was pipetted in a Biorad Poly-Prep® column and be rinsed four times with 1mL water. Furyl-modified oligosaccharide (1.25 mmol, 5 equiv.) was then added in 1mL water. After 16h in a rotary stirrer at 40°C the particles are rinsed with 10 mL of water. The resin is then stored in water with 0.1% sodium azide.
The ligation efficiency reached 85% conversion. The yield was determined by quantification of glucose released from the maltose-modified Sepharose particles after enzymatic treatment with a glucoamylase. Briefly, maltose-modified Sepharose particles (0.2 mL, 50% vol slurry in water) were placed in a Biorad Poly-Prep® column, rinsed 4 times with 0.1 M acetate buffer (pH 5) and incubated with a large excess of Glucoamylase AMGII (500µL of a 1 mg.mL -1 solution in the same buffer). The column was stirred 12h at 37°C. The amount of glucose in the supernatant was determined by the BCA reducing sugar assay.

Petrelli A., Samain E., Pradeau S., Halila S, & Fort S. (2017). Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels-Alder Reaction: Application to the Capture of Carbohydrate-Binding Proteins. Chembiochem : a European journal of chemical biology, 18(2).

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Purification of His-tagged BtuM Protein

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His-tagged BtuM for crystallisation was solubilised in buffer A (50 mM HEPES/NaOH pH 8, 300 mM NaCl, 0.05 mM cyano-Cbl, 1% n-dodecyl-β-maltoside (DDM) and 15 mM imidazole/HCl pH 8.5) for 45 min at 4 °C with gentle movement. Unsolubilized material was removed by centrifugation for 35 min at 219,373×g (average) and 4 °C. The supernatant was decanted into a poly-prep column (BioRad) containing 0.5 ml bed volume superflow Ni²⁺-NTA sepharose (GE healthcare) equilibrated with 20 column volumes (CV) buffer A containing additionally 3 mM dithiotreitol (DTT) and incubated for 1 h at 4 °C with gentle movement. Unbound protein was allowed to flow through and the column was washed twice with ten CV buffer A supplemented with 3 mM DTT and 0.35% n-nonyl-β-d-glucopyranoside (NG) and 60 mM or 90 mM imidazole/HCl pH 8.5. Bound protein was eluted from the column in four fractions of 0.5 ml (first)—0.7 ml (others) with buffer A supplemented with 3 mM DTT 0.35% NG and 350 mM imidazole/HCl pH 8.5. The sample was centrifuged for 5 min at 20,000×g and 4 °C to remove aggregates, and then loaded on a SD200 10/300 Increase SEC column (GE healthcare), which was equilibrated with 30 ml buffer B (50 mM HEPES/NaOH pH 8, 100 mM NaCl, 0.005 mM cyano-Cbl and 0.35% NG) and eluted in the same buffer while monitoring absorption at 280 and 361 nm.

Rempel S., Colucci E., de Gier J.W., Guskov A, & Slotboom D.J. (2018). Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM. Nature Communications, 9, 3038.

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Isolating Serpin-HP1 Complexes from Immune-Challenged Larvae

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Cell-free hemolymph samples (10 ml) from immune-challenged larvae were incubated with Micrococcus luteus (100 μg) for 30 min at room temperature to activate the SP-SPH system. To avoid melanization and protein crosslinking, 10 mM diethylthiocarbonate (a Cu²⁺ chelator that inhibits PO) and 1 mM 1-phenyl-2-thiourea (another PO inhibitor) were added to the reaction mixture. Rabbit antiserum against HP1 (4.8 ml) was coupled to 2.4 ml Protein A-Sepharose beads (Sigma) according to the manufacturer’s instructions. For isolating serpin-HP1 complexes, the plasma activated by M. luteus was mixed with the antibody-coupled beads for 8 h at 4°C with gentle agitation. The suspension was loaded into a Poly-Prep column (Bio-Rad) and then washed with 20 ml of 1 M NaCl and 20 ml of 10 mM sodium phosphate, pH 6.8 to remove unbound proteins. Bound proteins were eluted from the column with 50 mM glycine-HCl, pH 2.5. Fractions (0.5 ml each) were instantly neutralized with 50 μl, 1 M Tris-HCl, pH 8.0 in each collection tube. The elution fractions were separated by SDS-PAGE, followed by light staining with Coomassie Blue or immunoblot analysis.

He Y., Wang Y., Yang F, & Jiang H. (2017). Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses. Insect biochemistry and molecular biology, 84, 23-31.

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Extraction and Purification of Glycosaminoglycans

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Porcine mucosal heparin, heparan sulphate and chondroitin sulphate were purchased from Celsus (Cincinnati, OH, USA). Heparinases I, II, and III used in the large-scale depolymerization were obtained from Grampian Enzymes (Aberdeen, UK). Chondroitinase ABC was from IBEX (Montreal, QC, Canada). All other reagents used were of analytical grade. The alkaline protease mixture, alcalase, was from Novo (New York, NY, USA). Spectrapore dialysis membranes with a molecular cut-off weight of 1000 Da were from Spectrum Medical (Los Angeles, CA, USA). A small Polyprep column from BioRad (Watford, Hertfordshire, UK) was packed with 500 µL of Diethylaminoethyl DEAE Sephacel (GE-Healthcare, Visalia, CA, USA).

Khurshid C, & Pye D.A. (2018). Isolation and Composition Analysis of Bioactive Glycosaminoglycans from Whelk. Marine Drugs, 16(5), 171.

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Isolation and Analysis of Alkylated DNA from Reconstituted Nucleosomes

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Solutions of NCP reconstituted with alkylated DNA described above were concentrated to 30 μL using a 0.5 mL Amicon Ultra centrifugal filter with a molecular weight cutoff of 10 000 Da (4 °C). DNA associated with the reconstituted NCP and the remaining free DNA were separated by native PAGE (6%, acrylamide/bis(acrylamide), 59:1, 0.6 × TBE) using a running buffer of 0.2 × TBE and 200 V (1 h, 4 °C). The DNA species were detected by phosphorimagery and extracted from the gels by excision, maceration, and finally immersion in 2 mL of elution buffer (0.2 M NaCl, 1 mM EDTA) overnight at 4 °C. Solid material was removed by passage through a Bio-Rad Poly-Prep column and the eluant was lyophilized. The isolated DNA was incubated with proteinase K (1.6 U) for 15 min, heated with piperidine for cleavage at sites of guanine N7 alkyation, and analyzed as described above.

Byrne S.R., Yang K, & Rokita S.E. (2019). Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile. Chemical research in toxicology, 32(5), 917-925.

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Reconstitution of MexAB-OprM Membrane Protein Complex

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Reconstructed MexAB–OprM (~4 mg) was precipitated by mixing buffer D supplemented with 20% (v/v) PEG-3350 at a volume ratio of 1:2 and then centrifuging at 20,400 g for 30 min at 4 °C. After removal of the supernatant, 250 µL of 50 mM HEPES-K (pH 7.5) containing 20 mg of Amphipol A8–35 (Anatrace) was added to the pellet, and the sample was incubated at 4 °C for 4 h with gentle rotation. Subsequently, ~125 mg of Bio-Beads SM2 (Bio-Rad) was added to the sample, which was rotated at 4 °C overnight. The beads were removed with a poly-prep column (Bio-Rad). The sample was subjected to a Superose6 Increase 10/300 column (GE Healthcare) with 50 mM HEPES-K (pH 7.5). Peak fractions were concentrated with an Amicon Ultra-0.5 mL 100 K (Merck Millipore).

Tsutsumi K., Yonehara R., Ishizaka-Ikeda E., Miyazaki N., Maeda S., Iwasaki K., Nakagawa A, & Yamashita E. (2019). Structures of the wild-type MexAB–OprM tripartite pump reveal its complex formation and drug efflux mechanism. Nature Communications, 10, 1520.

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Purification of Recombinant Proteins

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All recombinant proteins were expressed in either BL21-CodonPlus (DE3)-RIPL or ArcticExpress (DE3) competent cells (Agilent Technologies) grown in LB medium overnight at 13–16°C. For GST-recombinants, the cells were lysed using a sonicator (FisherScientific) in GST-lysis buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) supplemented with lysozyme (500 μg/ml), 1% Triton-X100 and protease inhibitor cocktail (Sigma).
For His-tagged recombinants (IFE-4, PATR-1), the cells were lysed in His-lysis buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM Imidazole, 10% glycerol) supplemented with lysozyme, Triton-X100, and protease inhibitor cocktail. The protein was purified from cleared cell lysate using Ni-Sepharose 6 Fast Flow resin (GE Healthcare) in a Poly-Prep column (Bio-Rad). Following multiple washing steps with His-lysis buffer containing 60 mM imidazole, recombinants were eluted in His-lysis buffer containing 250 mM imidazole. Each fraction was analyzed by SDS-PAGE and Coomassie staining. Pure fractions were then concentrated using 50K centrifugal filter units (Amicon).

Mayya V.K., Flamand M.N., Lambert A.M., Jafarnejad S.M., Wohlschlegel J.A., Sonenberg N, & Duchaine T.F. (2021). microRNA-mediated translation repression through GYF-1 and IFE-4 in C. elegans development. Nucleic Acids Research, 49(9), 4803-4815.

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Transfection and Purification of MET Constructs

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pIRESneo-MET or pIRESneo-MET^Δ7–8 were transfected into HEK-293T or TOV-112D cells in 6-well culture dishes (Greiner Bio-One, Krëmsmunster, Austria) using Fugene HD transfection reagent (Promega, Fitchburg, WI, USA) according to the manufacturers’ instructions. After 48 h, cell monolayers were washed with PBS and cell extracts prepared in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology, CST, Danvers, MA, USA).
In separate experiments, pHLsec-MET^Δ7–8ED-BAPHIS or pHLsec-MET^ED-BAPHIS were cotransfected in a 1:1 ratio with the biotin-ligase expression construct pDISPLAY-BirA-ER (Addgene) in HEK293T cells and cells were cultured in the presence of 10 μM biotin (Sigma-Aldrich, St Louis, MO, USA). After 48 h, cytosolic extracts were made in RIPA buffer, and medium (1 ml) was mixed with 100 μl Ni–NTA Sepharose slurry (IBA, Goettingen, Germany). After a 1 h incubation at 4 °C, Ni-beads were washed with buffer (500 mM NaCl, 50 mM phosphate buffer, pH 7.4) and loaded onto a poly-prep column (Bio-Rad, Hercules, CA, USA). After washing off specifically bound proteins with 2 ml 10 mM imidazole, His-tagged an biotinylated MET extracellular domains were eluted with 0.5 ml 0.5 M imidazole, and dialysed o/n at 4 °C to 50 mM TRIS/150 mM NaCl pH 7.5.

Navis A.C., van Lith S.A., van Duijnhoven S.M., de Pooter M., Yetkin-Arik B., Wesseling P., Hendriks W.J., Venselaar H., Timmer M., van Cleef P., van Bergen en Henegouwen P., Best M.G., Wurdinger T.D., Tops B.B, & Leenders W.P. (2015). Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein. Acta Neuropathologica, 130(1), 131-144.

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Purification of Apo Proteins

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For purification of apo proteins, one aliquot of membrane vesicles was solubilised in 50 mM Tris-HCl pH 8.0, 300 mM KCl, 1% (v/v) n-dodecyl-β-D-maltoside (DDM) for 45 min at 4 °C. The insoluble fraction was removed by ultracentrifugation for 30 min at 265,000 × g at 4 °C and the soluble fraction was supplemented with 15 mM imidazole pH 8.0 and incubated with 0.5 mL Ni²⁺-sepharose resin (GE Healthcare) on a rocking platform at 4 °C for 1 h. Following this, the unbound fraction was allowed to flow through a Poly-prep column (Biorad) and the column was washed with 50 mM Tris-HCl pH 8.0, 300 mM KCl, 60 mM imidazole pH 8.0 and 0.15% (v/v) n-decyl-β-maltoside (DM). Then, the protein was eluted in the same buffer containing 500 mM imidazole pH 8.0. The eluted protein was further purified from imidazole by size exclusion chromatography on the Superdex 200 10/300 column (GE Healthcare) in the gel filtration buffer 10 mM HEPES-KOH, pH 8.0, 100 mM KCl and 0.15% DM.

Colucci E., Anshari Z.R., Patiño-Ruiz M.F., Nemchinova M., Whittaker J., Slotboom D.J, & Guskov A. (2023). Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6. Nature Communications, 14, 1799.

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Synthesis of Peptide-Based Crosslinker BDP-NHP

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The peptide-based cross-linker biotin–aspartate proline–N-hydroxyphthalimide (BDP–NHP) was synthesized using Fmoc chemistry as described (31 (link)) but with the use of Rink amide ProTide resin (CEM) instead of Fmoc-Gly-Wang resin. Amino acids were coupled to the resin in the following order: Lys(biotin)-Lys-Pro₂-Asp₂-succinate₂-N-hydroxyphthalimide (NHP)₂, using a Liberty Lite peptide synthesizer (CEM). Activated NHP esters were incorporated by incubating the resin containing the BDP molecule with a 12-fold molar excess of N-(trifluoroacetoxy)-phthalimide–NHP in pyridine. The reaction was carried out for 20 min at RT. The resin was then transferred to a PolyPrep column (Bio-Rad) and coupled to a vacuum flask. The resin was washed extensively with dimethylformamide followed by extensive washes with dichloromethane. The crosslinker was cleaved from the resin by incubation with 95% N-(trifluoroacetoxy), 2.5% dichloromethane, and 2.5% H₂O for 3 h at RT. The crosslinker was then precipitated in cold diethyl ether and washed extensively with fresh cold diethyl ether. The resulting pellet was dried by vacuum centrifugation. The resulting product was dissolved in dimethyl sulfoxide to a concentration of 200 mM, determined by UV–visible absorbance.

Bartolec T.K., Hamey J.J., Keller A., Chavez J.D., Bruce J.E, & Wilkins M.R. (2022). Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p. Molecular & Cellular Proteomics : MCP, 21(7), 100249.

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