Purified VP2 protein (600 µL), derived from the induction of 3 T75 flasks infected with recombinant MVA, was placed on the membrane of a centrifugal filter device (Amicon® Ultra 30K (Millipore, Burlington, MA, USA)) to remove the 3X FLAG peptide. Then, 100 µL of 100 mM NaCl was added to the purified VP2, before being centrifuged at 7000× g until the volume was less than 100 µL. One milliliter of anti-protease PBS with 10 mM NaCl was added to the membrane, and the column was centrifuged at 7000× g until a volume of 100 µL was obtained, which was then used to label the VP2 with an HRP Conjugation kit (Abcam, Cambridge, UK), following the manufacturer’s instructions.
Amicon ultra 30k
Manufactured by Merck Group
Sourced in Germany, United States
The Amicon Ultra 30K is a centrifugal filter device used for the concentration and desalting of protein samples. It is designed to retain molecules larger than 30 kilodaltons while allowing smaller molecules to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Aav helper free system, by Agilent Technologies (2 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Hrp conjugation kit, by Abcam (1 mentions)
His gravitrap talon columns, by GE Healthcare (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Sappβ, by Fortrea (1 mentions)
Sappα, by Fortrea (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Lifescope, by Thermo Fisher Scientific (1 mentions)
Solid5500, by Thermo Fisher Scientific (1 mentions)
Anti flag affinity gel, by Merck Group (1 mentions)
Incucyte sx5 live cell analysis system, by Sartorius (1 mentions)
Imidazole, by Merck Group (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
31 protocols using amicon ultra 30k
1
Purification and HRP Labeling of VP2 Protein
2
Purification of DESVand LIGVProteins
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The 1.5-kb NdeI-BamHI fragment carrying desV and the 1.9-kb NdeI-XhoI fragment carrying ligV from pT21-2832 and pLVH, respectively, were ligated into the corresponding sites of pET-16b. The resultant plasmids, pT16-desV and pT16-ligV, were independently introduced into E. coli BL21(DE3), and then the His tag-fused desV and ligV were expressed. For purification, cell extracts were applied to His GraviTrap TALON columns (GE Healthcare). Purified fractions were subjected to desalting and centrifugal filtration with Amicon Ultra 30k (Millipore), and stored at −30 °C until use. The purity of the preparations was examined by SDS–12% PAGE.
3
Quantifying APP Secretase Activity
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Cells were incubated with justicidin A for 8 h, and the media were concentrated using Amicon Ultra 30K centrifugal filters (Millipore). The concentrated media was immunoprecipitated with an APP antibody recognizing the N-terminus (abcam, MA, USA) and Protein G Agarose (Millipore). The immunoprecipitated samples were washed with PBS, and probed for sAPPα (Covance, NJ, USA) and sAPPβ (Covance) using Western blot.
4
Purification of Shiga Toxin
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The E. coli K12 strain that contained the plasmid that encoded for the stx of interest was grown overnight, sedimented by centrifugation (5,000×g), resuspended 40X in sonication buffer (50 mM NaPO4, 200 mM NaCl), and disrupted by sonication. The cell lysate was sedimented by centrifugation (20,000×g) and placed over the appropriate affinity column. After purification, Stx was dialyzed against PBS in Slide-A-Lyzer dialysis cassettes (Thermo Scientific) and concentrated (Millipore Amicon Ultra 30 K) according to manufacturer's instruction, if necessary. The protein concentrations of the Stx preparations were determined with a bicinchoninic acid (BCA) assay (Thermo Scientific).
5
Trio Whole Exome Sequencing for Variant Discovery
6
Purification of Recombinant proCTSA-6His
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The cell line stably producing proCTSA-6His (1 × 107 cells/mL) was cultured in serum-free Ham’s F-10 for 7 days, and then the supernatant was collected after being centrifuged at 1,500×g to remove floating cells as conditioned medium (CM). The pH of the supernatant was adjusted to below 7.5 and then was applied to the Ni-Sepharose column (6 Fast Flow, Cytiva). The column was washed with 20 mM sodium phosphate buffer (NaPB, pH7.5) containing 20 mM imidazole-HCl, and then the adsorbed proteins were eluted with NaPB (pH7.5) containing 500 mM imidazole-HCl. The eluate was concentrated, and the buffer composition was exchanged to PBS with Amicon Ultra 30k (30,000 MWCO; Millipore, Billerica, MA) and then stored at −30°C. The purity of the preparation was monitored by SDS-PAGE on a 12.5% polyacrylamide gel. Proteins were visualized with a PhastGel Blue R-350 (Cytiva). The purified proCTSA-6His was stored at −30°C before use.
7
Purification of Recombinant ERFE from HEK293T Cells
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Supernatants from HEK293T overexpressing FLAG-tagged ERFE were harvested after 5 days, supplemented with protease inhibitor cocktail and purified using an anti-FLAG affinity gel according to the manufacturer’s protocol (Sigma) and eluted with 150 μg/ml FLAG peptide (Sigma). FLAG peptide was then eliminated by filtration through Amicon Ultra 30K device (Millipore) and recombinant ERFE was resuspended in saline (0.9% NaCl). Protein concentration was determined by Coomassie Imperial Protein Stain (Pierce) and bicinchoninic acid protein assay (Pierce).
8
AAV-Mediated IL-37 Gene Delivery
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Vector construction, production, and in vivo delivery of adeno-associated virus (AAV) were performed based on the AAV helper-free system (Agilent). The recombinant adenoviral vector pAAV-IL37 was constructed by cloning the cDNA encoding region into pAAV-ITR. The vector pAAV-GFP encoding green fluorescence protein was used as a negative control. Recombinant AAVs were produced by HEK293 cells (ATCC) transfected with pAAV-ITR vectors together with pAAV-RC and pHelper plasmids, and then purified by discontinuous iodixanol gradient centrifugation. Purified recombinant AAVs were concentrated and desalted by centrifugation through Amicon Ultra 30K filters (Millipore). For in vivo delivery, recombinant AAVs equivalent to 1.0 × 1012 viral genome copies were delivered though mouse tail vein.
9
Abdominal Aortic Protein Analysis
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Abdominal aorta (between the diaphragm and the left renal artery) was minced and lysed in buffer containing 20 mM HEPES (pH: 7.9), 150 mM NaCl, 5 mM EDTA, 15% glycerol, 1% Triton X-100, a protease inhibitor cocktail and phosphatase inhibitor cocktail. Total cell lysate of the cultured cells was lysed in the same buffer. Cytosolic fraction of the cultured cells was obtained using ultracentrifuge. Culture media was concentrated using Amicon Ultra 30 K (Millipore). The proteins were fractioned using 8–10% SDS–polyacrylamide gel electrophoresis and analysed using immunoblotting. Densitometry analysis on the bands was calculated using ImageJ. The non-cropped blots for the representative images are displayed in Supplementary Fig. 7 .
10
Soluble ACE2 Binding Affinity Assay
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The soluble peptidase domain of human ACE2 (residues 19–615) was produced in a system of baculovirus/insect cells and purified at the IBV-Covid19-Pipeline (Institute of Biomedicine of Valencia-CSIC) as previously reported (Ginex et al. 2022 ). Purified protein was concentrated by centrifugal ultrafiltration using Amicon Ultra 30 K (Millipore) and quantified spectrophotometrically at 280 nm (mass extinction coefficient E 1 per cent: 21.9 g/l/cm). Ca. 5000 IU of each pseudotype were pre-incubated 1:1 with anti-VSV-G antibody and with serial dilutions of the soluble human ACE2 or a vehicle (PBS) control for 1 h at 37 °C, and then used to infect Vero E6-T cells seeded in 96-well plates. The quantity of GFP-positive cells at 24 hpi was measured using the Incucyte SX5 Live-Cell Analysis System (Sartorious) and the Incucyte analysis software. The percentage of infection inhibition, an indirect measure of binding affinity to ACE2, was calculated as 100× (GFP-positive cells with vehicle—GFP-positive cells with hACE2)/(GFP-positive cells with vehicle).
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