Coding sequences were cloned into a modified version of the Daedalus lentiviral transfer vector, which supported ligation independent cloning and expressed targets as a fusion with a variant of mIgG2a. Lentivirus production was essentially as described. Transduction of HEK-293F cells (Gibco) was carried out at a scale of 30 ml in Freestyle 293 Expression media (Gibco) and cultures were expanded to a 3 l production scale. Supernatant from 3 l culture was harvested by centrifugation (2000 × g, 10 min), 15 ml 1 M MES pH 6.5 and AEBSF (final concentration 20 µM) was added, and the supernatant swirled at 4˚C for 1 h with a 30 ml bed volume (bv) of His60 Ni-IDA resin (Clontech). Resin was poured into a gravity column, washed with 10 bv wash buffer (25 mM MES, pH 6.5, 150 mM NaCl, 10% glycerol, 50 mM Arginine-Cl and 5 mM imidazole)) and eluted with 5bv elution buffer (wash buffer with 100 mM Arginine-Cl and 0.5 M imidazole). Protein was concentrated using centrifugal units (Amicon Ultracel-10, Millipore), and applied to a gel filtration column (Sephacryl S200 26/60, GE lifesciences) equilibrated in 25 mM MES, pH 6.5, 150 mM NaCl, 10% glycerol, 100 mM Arg-Cl. Peak fractions were collected and concentrated by as above.
Amicon ultra cel 10
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra Cel-10 is a laboratory centrifugal filter device designed for the concentration and purification of macromolecules such as proteins, enzymes, and antibodies. The device utilizes a regenerated cellulose membrane with a molecular weight cut-off of 10 kDa to selectively retain the desired molecules while allowing smaller components to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression media, by Thermo Fisher Scientific (1 mentions)
Ab96233, by Abcam (1 mentions)
L4669, by Merck Group (1 mentions)
Image studio lite, by LI COR (1 mentions)
Jla20, by Merck Group (1 mentions)
Nb110 41568, by Novus Biologicals (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Human ifn beta duoset elisa, by R&D Systems (1 mentions)
Powerwave xs2, by Agilent Technologies (1 mentions)
5 protocols using amicon ultra cel 10
1
Lentiviral Protein Expression and Purification
2
Lysyl Oxidase Activity Assay in Cell Media
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Amplite™ Fluorimetric Lysyl Oxidase Assay Kit (ATT Bioquest®, Inc., Sunnyvale, CA, USA) was used to detect the activity of lysyl oxidase in the media of N1 and WM793 cells. After growing for two days in normal serum containing media the cells were washed and transferred to serum free media for 18 h. The conditioned media were collected, and secreted proteins were concentrated using Amicon Ultra Cel-10 centrifugal filters (MWCO 10.000; Millipore, Bedford, MA, USA), as previously described [94 (link)]. The lysyl oxidase activity in the concentrated media was measured in the presence of 0, 250, or 500 μM ΒAPN and 0.1% albumin. Recombinant LOXL2, rhLOXL2 (Bio-techne Ltd, Abingdon, UK), was used as a positive control.
3
Protein Concentration and Analysis
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Secreted proteins were first concentrated using Amicon Ultra Cel-10 centrifugal filters (MWCO 10.000; Millipore, Bedford, MA, USA) as previously described [94 (link)]. Cellular lysates or secreted proteins were resolved by 10% SDS-PAGE, and the proteins were electrophoretically transferred onto nitrocellulose membrane (Bio-Rad, Herwles, Hercules, CA, USA) and analyzed as previously [24 (link)]. Secreted and cellular protein bands of human melanoma cells and fibroblasts were quantified using Image Studio Lite (LI-COR Biotechnology, Lincoln, NE, USA). A rabbit polyclonal antibody recognizing the C-terminal domain of LOX (L4669; Sigma-Aldrich), a rabbit polyclonal LOX propeptide antibody to LOX-PP (NB110-41568; Novus Biologicals, Littleton, CO, USA), and a rabbit polyclonal antibody to LOXL2 (ab96233; Abcam, Cambridge, UK) were used to detect the respective proteins. Secreted protein samples were verified to contain comparable amounts of total proteins by silver staining. Mouse monoclonal antibodies to alpha-tubulin (DM1A; Abcam) and actin (JLA20; Merck, Millipore, Billerica, MA, USA) were used as loading controls for cellular proteins.
4
Enzymatic Conversion of Coffee Waste
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Coffee waste was obtained by grinding commercial coffee beans in a hand mill and mixing them with boiled water, followed by filtration. The residues were collected and dried. A portion of the material was further ground to a powder at 1,200 rpm for 1 min using Auto-mill TK-AM5 (Tokken Inc., Chiba, Japan).
Recombinant TtMan5A (0.4 mg) was incubated with the dried coffee waste (15 mg) in McIlvaine buffer (pH 4.5) at 50 °C for up to 69 h in a final reaction volume of 1 mL. The reaction mixture was filtrated using Amicon Ultracel-10 (Merck Millipore Co.). Saccharification ability of TtMan5A was evaluated by measuring the reducing sugars of the flow-through fractions and by analyzing the sugar composition of the residues. The hydrolysis products were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (DX-500; Dionex Corp., Sunnyvale, CA, USA). β-1,4-Mannooligosaccharides and galacto-mannooligosaccharides, such as 61-mono-α-D-galactopyranosyl-β-1,4-mannobiose (GM2) and 61-mono-α-D-galactopyranosyl-β-1,4-mannotriose (GM3), from Megazyme International were used as standards. Enzyme-treated and -untreated residues were washed three times with distilled water and freeze-dried. Sugar composition of the residues was analyzed using trifluoroacetic acid (TFA) hydrolysis and HPAEC-PAD as previously described.26) 27) (link)
Recombinant TtMan5A (0.4 mg) was incubated with the dried coffee waste (15 mg) in McIlvaine buffer (pH 4.5) at 50 °C for up to 69 h in a final reaction volume of 1 mL. The reaction mixture was filtrated using Amicon Ultracel-10 (Merck Millipore Co.). Saccharification ability of TtMan5A was evaluated by measuring the reducing sugars of the flow-through fractions and by analyzing the sugar composition of the residues. The hydrolysis products were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (DX-500; Dionex Corp., Sunnyvale, CA, USA). β-1,4-Mannooligosaccharides and galacto-mannooligosaccharides, such as 61-mono-α-D-galactopyranosyl-β-1,4-mannobiose (GM2) and 61-mono-α-D-galactopyranosyl-β-1,4-mannotriose (GM3), from Megazyme International were used as standards. Enzyme-treated and -untreated residues were washed three times with distilled water and freeze-dried. Sugar composition of the residues was analyzed using trifluoroacetic acid (TFA) hydrolysis and HPAEC-PAD as previously described.26) 27) (link)
5
Quantitative IFN-β Immunoassay Protocol
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Growth medium supernatants were centrifuged to exclude floating cells. Resulting supernatants were 20X up-concentrated by centrifuge filtering through 10 kDa cut-off columns (Amicon Ultracel-10, Merck). ELISA (Human IFN-beta DuoSET ELISA, R&D Systems) was conducted according to supplier’s protocol. Optical density was measured at 450 nm with pathlength correction at 540 nm in a microplate spectrophotometer (PowerWave XS2, BioTek) coupled to the Gen5 software v2.09.1. IFN-β standards were included in all experiments, and a best-fitting 2nd degree polynomial function was used for calculation of measured IFN-β in the samples.
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