Five mg of lyophilized PEP04314 (0.71 µmol, Affibody AB, Solna, Sweden) was added to 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.5), and the solution was added to a reaction vial containing 0.4 mg (0.72 µmol) of (±)-H3RESCA-maleimide or maleimide-mono-amide-NOTA (Macrocyclics, Plano, Tx, USA). The reaction mixture was kept at room temperature for 90 min before being transferred to an ultracel 3K centrifugal filter (Merck) containing 3 mL of 0.1 M ammonium acetate buffer (pH 4) and centrifuged at 4000 rpm for 90 min. The flow-through was discarded, and 4 mL of fresh 0.1 M ammonium acetate buffer (pH 4) was added. The filter was then centrifuged again for 90 min, and the flow-through was discarded. Purified (±)-H3RESCA-PEP04314 or H2NOTA-PEP04314 was collected via reverse spin in 1 mL of 0.1 M ammonium acetate buffer (pH 4.5), and stored at -70 °C in 100 µL aliquots prior to use. Purity of the final product was determined via a Waters Acquity LC/MS system equipped with a Waters Xselect CSH C18 column (250 mm ×10 mm, 130 Å) at a flow rate of 5 mL/min using a gradient of EtOH (10-40% over 15 min) and 0.1% formic acid.
Ultracel 3k centrifugal filter
Manufactured by Merck Group
Sourced in Ireland
Ultracel®-3K centrifugal filters are a type of laboratory equipment used for sample preparation and purification. These filters feature a molecular weight cut-off of 3,000 Daltons, allowing for the efficient separation and concentration of molecules based on their size. The core function of Ultracel®-3K centrifugal filters is to facilitate the rapid and precise filtration and concentration of biological samples, such as proteins, peptides, and other macromolecules.
Automatically generated - may contain errors
5 protocols using ultracel 3k centrifugal filter
1
Radiolabeling of Affibody Molecules
2
Fractionation of Low and High Molecular Weight Polyphenol Extracts
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PPE (500 μL) was applied to an Ultracel-3K centrifugal filter device (Merck Millipore) and centrifuged at 16,100×g at 4 °C for 60 min. The filtrates were used as a LMW fraction of PPE. To recover the residues on the filter, the filter devices were placed upside down in a new centrifuge tube and centrifuged at 1000×g at 4 °C for 5 min. The residues were reconstituted with 500 μL of phosphate-buffered saline (PBS) and used as a HMW fraction of PPE.
3
Deglycosylation of Thermostable Lectin
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TLA was heat-inactivated by incubation for 15 min at 95°C (hiTLA) and deglycosylation (dgTLA) was performed by sodium metaperiodate treatment according to a modified protocol (36 (link)). Briefly, 3 mg TLA (1 mg/ml in PBS) were mixed with 3 ml 100 mM sodium metaperiodate in 100 mM acetate buffer, at pH = 4.5. TLA was incubated at 37°C for 30 min in the dark. The oxidation reaction was stopped with 300 ml 0.5 x PBS. The volume was then reduced to 5 ml by centrifugation at 2,900 x g at 4°C using Ultracel®-3K centrifugal filters (Amicon® Ultra-15; Merck Millipore). Volume was further decreased with a Vacuum Concentrator Centrifuge (Univapo 150H; Uniequip). The protein concentration was assessed as above and modification of glycan moieties was verified with a Western blot using biotinylated Concanavalin A (ConA) as described below.
4
Protein Detection from Cell Supernatant
5
Quantifying TNFα-induced CCL2 Secretion
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WT and 26A PC3U cells were cultured with or without TNFα stimulation for 6 h. The conditioned medium was collected, centrifuged at 300 × g for 10 min, and then centrifuged with Ultracel-3K Centrifugal filters (Merck Millipore Ltd, Tullagreen Carrigtwohill Co, Ireland) at 4000 × g for 30 min. Secretion of CCL2 into the medium was determined by using the DuoSet ELISA -Human CCL2/MCP-1 kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocols. Optical density (O.D.) at 450 nm was measured in a 96-well plate by Multiscan FC Microplate Photometer (Thermo Scientific).
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