Ultrafiltration membrane

Manufactured by GE Healthcare

Sourced in United Kingdom, Sweden, Japan

The Ultrafiltration membrane is a laboratory equipment designed for the separation and concentration of molecules based on their size. It functions by allowing small molecules to pass through the membrane while retaining larger molecules, effectively concentrating the desired components in the sample.

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Lab products found in correlation

Superdex 75 column, by Cytiva (1 mentions) Detoxi gel endotoxin removing gel, by Thermo Fisher Scientific (1 mentions) Qproteome bacterial protein prep kit, by Qiagen (1 mentions)

3 protocols using ultrafiltration membrane

Protein Fractionation and Endotoxin Removal

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The TE was fractionated by size exclusion fast protein liquid chromatography (FPLC) on an ÄKTAFPLC Purifier UPC 10 (GE Healthcare Life Science, Sweden) system with a Superdex 200 HR 10/30 or Superdex 75 column (Amersham Bioscience, USA) in the PBS buffer (pH 7.4) and the fractions (25–30) were then collected.
To obtain F<10 kDa, TE 20 ml (6 mg/ml) was passed through an ultrafiltration membrane (GE Health Care, UK) with a molecular weight cut-off (MWCO) of 10 kDa in a microcentrifuge. The recombinant of Fh-KTM (rFh-KTM) was ordered by Shanghai Apeptide Co. Ltd. (Shangai, China), according to a sequence described by Bozas et. al. [17] (link). Endotoxin contamination was removed in both antigens by a detoxi-gel endotoxin removing gel (Pierce Biotechnology, Rockford USA), with the endotoxin level found being similar to those of the background and complete RPMI 1640 medium.

Falcón C.R., Masih D., Gatti G., Sanchez M.C., Motrán C.C, & Cervi L. (2014). Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses. PLoS ONE, 9(12), e114505.

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Isolation and Purification of Fibrinogenolytic Enzymes from L. rubellus Extract

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Pure active components of DLBS1033P were obtained from DLBS1033, the L. rubellus crude extract from West Java (Indonesia). The DLBS1033 was extracted using purified water, followed by continuous centrifugation (7780 Kubota, Japan) at 6,000xg for 15 min. The supernatant was then filtered using a microfiltration membrane of 0.1 μm (GE, Sweden) and concentrated using ultrafiltration membrane (cut off 10 kDa) (GE, Sweden). The concentrate was purified by Fast Performance Liquid Chromatography AKTA Purifier (GE Healthcare, Sweden) using ion-exchange chromatography, HiPrep DEAE (diethylaminoethyl) FF column (GE, Sweden). The column was equilibrated with a 20 mM phosphate buffer (pH 7.5). The adsorbed proteins were eluted with a stepwise gradient of 0.25 M NaCl (25, 55, and 85%) in the same buffer at a 5 mL/min flow rate. Each protein peak eluted from the 25 and 85% gradients exhibiting fibrinogenolytic activity was harvested for further analysis.

Stephani L., Rahayu P., Retnoningrum D., Suhartono M.T., Rachmawati H, & Tjandrawinata R.R. (2023). Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus. Proteome Science, 21, 8.

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Recombinant Protein Purification from E. coli

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E. coli BL21 (DE3) cells harboring appropriate plasmids were grown in Luria-Bertani broth containing ampicillin (100 mg/mL) at 30 °C until OD 600 reached 0.5. Protein expression was then induced by addition of IPTG (1 mM) followed by incubation with vigorous shaking at 30 °C for 4 hr. Cells were collected by centrifugation (6000 g at 4 °C for 20 min), washed three times with 20 mM sodium phosphate buffer (pH 7.4) and protease inhibitor (phenylmethanesulfonyl fluoride, leupeptin and pepstatin), and resuspended in lysis buffer (Qproteome bacterial protein prep kit; Qiagen, Hilden, Germany). Supernatant obtained by centrifugation (14,000 g at 4 °C for 30 min) was concentrated using an ultrafiltration membrane (GE Healthcare Japan, Tokyo, Japan), dialyzed against sodium phosphate buffer (20 mM, pH 7.4) containing 20 mM imidazole, and loaded into his-tag affinity resin (GE Healthcare Japan). After the column had been washed with washing buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) and 20-100 mM imidazole, recombinant proteins were eluted with the same buffer containing 500 mM imidazole. Eluted proteins were concentrated using an ultrafiltration membrane and analyzed by 5-20% gradient SDS-PAGE. Protein concentrations were determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA).

Otsuka R., Imai S., Murata T., Nomura Y., Okamoto M., Tsumori H., Kakuta E., Hanada N, & Momoi Y. (2015). Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation. Microbiology and immunology, 59(1).

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