The soluble anti-spike nanobody-IFN-β recombinant proteins were produced in 293T cells using transient transfection and purified using anti-DYKDDDDK tag antibody beads (FUJIFILM WAKO) as previously described [25 (link)]. Briefly, The soluble anti-spike nanobody-IFN-β recombinant proteins were captured with anti-FLAG tag antibody beads from the supernatant, washed, and competitively eluted using 150ug/ml FLAG peptide. The eluates were concentrated with Amicon Ultra centrifugal filter 3K (Millipore, Germany) to remove the residual FLAG peptide. LILRB3-FLAG was used for the control recombinant protein.
Amicon ultra centrifugal filter 3k
Manufactured by Merck Group
Sourced in Germany
The Amicon Ultra centrifugal filter 3K is a laboratory equipment product designed for rapid sample concentration and buffer exchange. It features a 3,000 molecular weight cutoff membrane that allows small molecules to pass through while retaining larger molecules of interest.
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Lab products found in correlation
4 protocols using amicon ultra centrifugal filter 3k
1
Purification of Spike-IFN-β Nanobody
2
Evaluating ERRα Impact on TGF-β
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The effects of ERRα on the expression of TGF-β in medium of EC cells were evaluated by use of a TGF-β ELISA kit (Invitrogen, Basel, Switzerland). Briefly, supernatants of 2 × 106 cells treated with or without XCT-790, a potent and selective inverse agonist ligand of ERRα, for 24 h were collected and concentrated using an Amicon Ultra centrifugal filter (3K) (Millipore, Temecula, CA). Then levels of TGF-β in medium were measured by use of the kit according to the manufacturer's instructions.
3
Lactobacilli Modulate Candida-induced Cytotoxicity
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Lactobacilli were grown in MRS broth for 24 h at 37°C. The MRS-SN was prepared by centrifuging the culture at 4700 g for 5 min at 4°C, filtering through 0.22 µm filters (Millipore) and rebuffering against KBM medium within a 3K Amicon Ultra centrifugal filter device (Millipore) (final pH 7.3) according to the manufacturer's instructions. Moreover, IECs differentiated in 6-well plates were colonized with lactobacilli for 18 h. The culture supernatant (Pre-Inc. SN) was filtered through 0.22 µm filters (Millipore). Both supernatants were stored at 4°C until use in experiments the same day (see Table S1 ). Host cell damage induced by C. albicans infection in the presence of lactobacilli supernatants was measured 24 h post-infection by quantification of LDH (for format and volume see Table S1 ).
4
Proteomic Analysis of KARS Overexpression
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The volume of supernatants from HEK 293T (3 mg protein), KARSoe (3 mg protein) and streptavidin affinity purification from KARSoe cells (denoted as KARSoe-AP afterwards) (200 μg protein) was adjusted to 500 μL using NETN lysis buffer. These samples were loaded onto a Superdex 200 10/300 GL column in an ÄKTA FPLC system (GE healthcare, Uppsala, Sweden), and eluted with PBS buffer at an optimal flow rate of 0.5 mL/min. The eluate was monitored by UV absorption at 280 nm, collected in 500 μl fractions, and exchanged with 50 mM Tris-Cl (pH 7.5) plus 6 M urea employing 3K Amicon ultra centrifugal filter (Millipore). The amount of protein in each fraction was measured by BCA protein assay.
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