Centriprep

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, Ireland

The Centriprep is a centrifugal concentration device used for the rapid concentration and desalting of macromolecular solutions. It utilizes a membrane-based filtration process to separate and concentrate the desired components from the original solution.

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19 protocols using centriprep

Purification of Recombinant Proteins from E. coli

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The E. coli host Shuffle^® T7 (New England Biolabs, Inc., Ipswich, MA, USA), was transformed with a cloned expression vector. The cells were cultured in 500 mL of LB medium containing 50 μg/mL of ampicillin at 30 °C until the optical density (OD) at 600 nm was 0.5. Proteins were overexpressed by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) treatment at 20 °C. After 5 h of reactions, cells were harvested by centrifugation (3000× g for 20 min). Harvested cells were lysed via sonification in a lysis buffer of 50 mM Tris-HCl, 200 or 300 mM NaCl, and 10% glycerol at pH 7.0. The cell lysates were filtered through a polyether-sulfone syringe filter with a 0.45 μm pore size (Hyundai Micro Co., Ltd., Seoul, Republic of Korea) after centrifugation at 16,000× g for 10 min and purification via Ni-affinity chromatography (Bio-Scale™ Mini Nuvia™ IMAC Cartridges; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The purified samples were concentrated with Centriprep™ (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). Protein samples were further separated by their size through size exclusion chromatography (SEC) with a Superdex™ 200 Increase 10/300 GL column (GE Healthcare, Uppsala, Sweden). After SEC, eluted fractions of the expected peak were pooled and concentrated with Centriprep™ (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland).

Ahn J., Yu J.E., Kim H., Sung J., Han G., Sohn M.H, & Seong B.L. (2023). AB5-Type Toxin as a Pentameric Scaffold in Recombinant Vaccines against the Japanese Encephalitis Virus. Toxins, 15(7), 425.

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Purification of Isotopically Labeled P51G CV-N Protein

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P51G CV-N, labeled with ¹⁵N or ¹³C/¹⁵N was expressed as described earlier.³² The protein was prepared from the soluble fraction after cell lysis by sonication. The cell debris was removed by centrifugation and the supernatant was dialyzed against 20 mM Tris-HCl buffer (pH 8.5), loaded onto a Q(HP) column (GE Healthcare) and protein was eluted using a linear gradient of 0–1 M NaCl. Protein-containing fractions were concentrated and further purified by gel filtration on a Superdex 75 column (GE Healthcare) in 50 mM Tris-HCl, 100 mM NaCl (pH 8.5). Fractions containing pure P51G CV-N were collected and concentrated using Centriprep and Amicon devices (Millipore). Samples for NMR were buffer-exchanged into 10 mM phosphate buffer, 3 mM NaN₃, 95/5% H₂O/D₂O (pH 6.6).

Nestor G., Anderson T., Oscarson S, & Gronenborn A.M. (2017). Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy. Journal of the American Chemical Society, 139(17), 6210-6216.

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Purification of Flavin-Bound sp-IDI-2

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E. coli M15 (p-sp-IDI-2) was grown at 37 °C to OD₆₀₀ ~0.6. Isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.4 mM) and flavin mononucleotide (FMN, final concentration 40 mg/L) were added and incubation was continued for 23 h at room temperature. Cells were pelleted and stored at −80 °C until needed. Frozen cells were thawed in binding buffer (20 mM sodium phosphate, pH 7.4, containing 500 mM NaCl and 20 mM imidazole) and disrupted with a Sonifier Cell Disruptor (8 cycles of 30 s, on ice).
sp-IDI-2 was purified by nickel-ion affinity chromatography on a HisTrap FF crude column (GE Heathcare) eluted with 50 mM Tris-HCl buffer, pH 7.4, containing 20–500 mM imidazole. Fractions of sp-IDI2 were pooled, concentrated with a 30 kD MWCO filter (Centriprep, Millipore) and dialyzed three times against 1 L of 10 mM Tris-HCl buffer, pH 8, containing 20% glycerol. This procedure gave sp-IDI-2 with a substoichiometric amount of bound FMN. An additional wash step with 2 M KBr in binding buffer just before elution with imidazole gave deflavinated apo-sp-IDI-2.^{[21 (link)]} Protein concentrations were determined by the BCA assay (Pierce).

de Ruyck J., Janczak M.W., Neti S.S., Rothman S.C., Schubert H.L., Cornish R.M., Matagne A., Wouters J, & Poulter C.D. (2014). Determination of kinetics and crystal structure of a novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae. Chembiochem : a European journal of chemical biology, 15(10), 1452-1458.

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Fusion Protein Generation and AP Staining

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To generate fusion proteins, cDNAs corresponding to the ectodomain were introduced into the APtag-5 (GenHunter Corporation) of AP fusion vectors. DNA fragments of the ectodomain were obtained by PCR. This enables the ectodomain to be fused to the N terminus of AP. AP fusion proteins were produced in the transfected HEK293T cells and concentrated using Centriprep (Millipore). Fresh-frozen OB sections were postfixed at −20 °C for 20 min in 100% methanol. AP staining was performed following the standard method^{7 (link), 9 (link), 17 (link)}.

Inoue N., Nishizumi H., Naritsuka H., Kiyonari H, & Sakano H. (2018). Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation. Nature Communications, 9, 1842.

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Detecting KIT Exon 10 SNP Using CAST-PCR and Sequencing

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The detection of the SNP was performed by Competitive Allele-Specific Taqman® PCR technology provided by Applied Biosystems®, using the method extensively described by Dufresne et al. [15 (link)]. We both performed a CAST®-PCR and a classical sequencing in 13 cases. Polymerase chain reaction (PCR) was used to amplify KIT exon 10. The primers used were designed by Primer Express and generated by Beckman Coulter Genomics: 5’-GTA-ATC-GTA-GCT-GGC-ATG-ATG-TG-3’ (sense) and 3’-AAC-CAT-TTA-TTT-GTT-CTC-TCT-CCA-GAG-5’ (antisense). PCR was carried out using Taq DNA polymerase (Transgenomic, Omaha, NE, USA) with the following cycle conditions: denaturation at 95 °C, followed by 10 cycles at decreasing temperatures between 66 and 61 °C with a decrement of 0.5 °C per cycle, and additional extension at 72 °C. The primers used for sequencing were the same as those used for amplification. Before sequencing, PCR products were purified using Centriprep centrifugal filter device (Millipore, Billerica, MA, USA). The purified PCR product was then subjected to automated sequencing performed by Beckman Coulter Genomics. Results of that double detection of the SNP, by sequencing and CAST®-PCR, were always consistent.

Brahmi M., Alberti L., Dufresne A., Ray-Coquard I., Cassier P., Meeus P., Decouvelaere A.V., Ranchère-Vince D, & Blay J.Y. (2015). KIT exon 10 variant (c.1621 A > C) single nucleotide polymorphism as predictor of GIST patient outcome. BMC Cancer, 15, 780.

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Cytokine Profiling of Conditioned Media

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Conditioned medium (CM) was prepared from 5T33MMvt cells or HMCL (RPMI8226, OPM2, LP1) cultured for 48 h in RPMI1640 medium (10% FCS) at a concentration of 10⁶/mL. For experiments, conditioned medium was diluted ½ in fresh RPMI1640 medium (10% FCS). Ten times concentrated conditioned medium with Centriprep (Millipore, Billerica, MA, USA) was used to detect cytokines with the mouse cytokine array panel A (R&D sytems, Minneapolis, MN, USA) according to manufacturer's instructions.

De Veirman K., Van Ginderachter J.A., Lub S., De Beule N., Thielemans K., Bautmans I., Oyajobi B.O., De Bruyne E., Menu E., Lemaire M., Van Riet I., Vanderkerken K, & Van Valckenborgh E. (2015). Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells. Oncotarget, 6(12), 10532-10547.

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Recombinant Expression of Trypanosome Proteins

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The genes encoding TcVSP1 and TbVSP1 were derived as described in our previous studies(7 (link), 8 (link)). Genes were amplified by polymerase chain reaction (PCR) with the primers listed in Table S4 and then cloned into the pET46 Ek/LIC vector (Novagen, Madison, Wisconsin, USA). Recombinant plasmids were verified by sequencing and transformed into E. coli BL21(DE3). Recombinant E. coli cells were grown at 37 °C to an OD₆₀₀ of 0.6. Protein expression was induced at 16 °C for 24 h by adding 1 mM IPTG. Cells were harvested by centrifugation at 5000×g at 4 °C for 20 min. Cell pellets were then resuspended in lysis buffer consisting of 25 mM Tris, pH 7.5, 150 mM NaCl, and 20 mM imidazole, then disrupted with a French press (GuangZhou JuNeng Biology and Technology Co. Ltd, Guangzhou, China). The supernatant was collected by centrifugation and purified by FPLC using Ni-NTA and DEAE columns (GE Healthcare, Uppsala, Sweden). The purified proteins were concentrated using a Centriprep (Millipore, Darmstadt, Germany) and purity was checked by SDS-PAGE. All purification procedures were performed at 4 °C.

Yang Y., Ko T.P., Chen C.C., Huang G., Zheng Y., Liu W., Wang I., Ho M.R., Danny Hsu S.T., O’Dowd B., Huff H.C., Huang C.H., Docampo R., Oldfield E, & Guo R.T. (2016). Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Anti-Parasitic Drug Targets. ACS chemical biology, 11(5), 1362-1371.

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IgG Purification from Sera

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Sera from patients and control subjects were clarified by centrifugation at 1500g for 20 min (500 μl) and filtration through 0.45-μm filters (Millipore, County Cork, Ireland). IgG fractions were prepared using a MAb Trap Kit (GE Healthcare, Tokyo, Japan) according to the manufacturer’s instructions. Purified IgG fractions were concentrated using Centriprep centrifugal filters (Millipore) and stored at 4°C until required.

Hara A., Furuichi K., Koshino A., Yasuda H., Tran T.T., Iwata Y., Sakai N., Shimizu M., Kaneko S., Nakamura H, & Wada T. (2017). Clinical and Pathological Significance of Autoantibodies to Erythropoietin Receptor in Type 2 Diabetic Patients With CKD. Kidney International Reports, 3(1), 133-141.

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HBV Infection Assay in Cell Lines

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The Hep38.7-Tet cell culture medium was used as an HBV inoculum (genotype D). Hep38.7-Tet cells were seeded and cultivated for 7 days in the absence of tetracycline. The supernatant was concentrated using a Centriprep (Milipore) to 1/20 of its original volume. Cells were seeded on collagen-coated 6-well plates at 2 × 10⁵ cells per well with DMEM including G418 overnight. The medium was then replaced with dHCGM. NmcHepG2 cells were next infected with condensed HBV and polyethylene glycol 8000 (4%). The following day, the medium was replaced with dHCGM containing the test compounds.
PXB cells were infected with serum of an HBV-infected chimeric mouse (genotype C) in the presence of polyethylene glycol 8000 (4%; 5 Geq/cell) containing dHCGM. The following day, the medium was replaced with dHCGM containing the drugs.

Takeuchi F., Ikeda S., Tsukamoto Y., Iwasawa Y., Qihao C., Otakaki Y., Ryota O., Yao W.L., Narita R., Makoto H., Watashi K., Wakita T., Takeuchi K., Chayama K., Kogure A., Kato H, & Fujita T. (2019). Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor. PLoS ONE, 14(2), e0212233.

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Comparison of Viral Extraction Methods

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Samples of coastal water (1 L) were analyzed by two methods based on negative-charged membrane filtration (MF) (Katayama et al., 2002 (link)) and FeCl₃ flocculation (FF) (John et al., 2011 (link)). For method MF, coastal water samples were directly filtered on a negative-charged HA-type membrane with a 47 mm diameter and 0.45 μm pores (Millipore, Burlington, MA, USA) placed on a vacuum sterile bottle. Filters were rinsed with 100 mL of 0.5 mM H₂SO₄ (pH 3) prior to viral elution with 1 mM NaOH (pH 10.5). After pH neutralization, 10 mL of viral suspension were concentrated using a 50 kda Centriprep ultrafiltration device (Millipore) to obtain 2 mL of viral concentrate. In parallel, for method FF, 200 μL of 10 g/L FeCL₃ solution was added to the filtrate from method MF (kept at 4 °C), and incubated 2 h at 10 °C under gentle agitation, in the dark. A flocculate was then collected on a 0.8 μm pore-size polycarbonate filter (Whatman, Maidstone, UK). Virus resuspension was achieved with 2 mL of ascorbate-oxalate–EDTA buffer during a 30 min incubation at 4 °C under agitation. Viral suspensions (method FF) and concentrates (method MF) were extracted using the NucliSens kit (bioMérieux, Lyon, France) with 10 mL of lysis buffer and 140 L of magnetic silica, and eluted in 100 μL of the kit's elution buffer.

Desdouits M., Piquet J.C., Wacrenier C., Le Mennec C., Parnaudeau S., Jousse S., Rocq S., Bigault L., Contrant M., Garry P., Chavanon F., Gabellec R., Lamort L., Lebrun L., Le Gall P., Meteigner C., Schmitt A., Seugnet J.L., Serais O., Peltier C., Bressolette-Bodin C., Blanchard Y, & Le Guyader F.S. (2021). Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?. The Science of the Total Environment, 778, 146270.

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