Unless otherwise noted, all steps were done at 4° C. Cells were resuspended in 20 mM sodium phosphate buffer pH 7.5, 500 mM NaCl, 5 mM MgCl2, 20 mM imidazole, 10 % (v/v) glycerol, 0.25 mM PMSF (lysis buffer), supplemented with 1 tablet complete EDTA-free (Roche, Switzerland) and 1 spatula tip DNaseI and RNaseA, lysed by two passages through a cell homogenizer (PandaPlus 2000, GEA Niro Soavi, Italy) at 1000 bar and subsequently centrifuged for 1 h at 50,000 × g. The supernatant was loaded on a 5 ml Ni-Sepharose HP column (GE Healthcare, USA), equilibrated with 20 mM sodium phosphate buffer pH 7.5, 500 mM NaCl, 20 mM imidazole, 10 % (v/v) glycerol, 0.25 mM PMSF (NiNTA buffer). The column was washed with 54 mM imidazole in NiNTA buffer and the mNG dimer was eluted with NiNTA buffer containing 260 mM imidazole. The eluate was concentrated with an Amicon Ultra 15 (Merck, Germany) ultrafiltration device (10 kDa cutoff) and loaded on a self-packed XK26/40 Sephacryl S300 size exclusion column (GE-Healthcare, USA) equilibrated with 20 mM sodium phosphate buffer pH 7.5, 150 mM NaCl, 10 % (v/v) glycerol, 0.25 mM PMSF. The eluted protein was concentrated via an Amicon Ultra 15 (Merck, Germany) ultrafiltration device (10 kDa cutoff), aliquoted, flash-frozen in liquid nitrogen and stored at −80°C.
Amicon ultra 15
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, United Kingdom, France, Switzerland, Japan, India
The Amicon Ultra-15 is a centrifugal filter device designed for the concentration and purification of macromolecules such as proteins, peptides, and nucleic acids. It features a regenerated cellulose membrane with a specific molecular weight cut-off, enabling the selective retention of the target analyte while allowing smaller molecules to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Bca protein assay, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Exoquick tc, by System Biosciences (6 mentions)
Ni nta resin, by Qiagen (5 mentions)
Histrap hp column, by GE Healthcare (5 mentions)
Ultracel 3 membrane, by Merck Group (5 mentions)
Superdex 200, by GE Healthcare (5 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (4 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (4 mentions)
0.22 μm filter, by Merck Group (4 mentions)
Nmri mice, by Charles River Laboratories (3 mentions)
Incomplete freund s adjuvant, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Glycoworks rapifluor ms n glycan kit, by Waters Corporation (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Nanoassemblr benchtop, by Precision Nanosystems (3 mentions)
639 protocols using amicon ultra 15
1
Purification of Monomeric Neon Green Protein
2
Purification of Keap1 Protein
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The cell pellets were treated with BugBuster HT Protein Extraction Reagent (Novagen). The soluble fraction was applied onto a His-Trap HP column (GE healthcare) equilibrated with the binding buffer [500 mM sodium chloride, 20 mM sodium phosphate buffer pH 7.4]. The column was washed with 2% Buffer A [1 M imidazole, 500 mM sodium chloride, 20 mM sodium phosphate buffer pH 7.4], and the His-tagged protein was eluted with 30% Buffer A. The eluate was concentrated by ultrafiltration using an Amicon Ultra-15 (Millipore, 10,000 cut-off) and applied onto a HiLoad 16/60 Superdex 200 column (GE healthcare Amersham) equilibrated with Buffer B [150 mM sodium chloride, 20 mM DTT (dithiothreitol), 20 mM Tris–HCl pH 8.3]. Peak fractions containing the protein were pooled, diluted with Buffer C [20 mM DTT, 25 mM Tris–HCl pH 8.0], and applied onto a HiTrap Q FF column (GE healthcare) equilibrated with 10% Buffer D [1 M sodium chloride, 20 mM DTT, 25 mM Tris–HCl pH 8.0]. The protein was eluted with linear gradient of 10–40% Buffer D. The purity of the protein sample was evaluated by SDS–PAGE, which showed a single band on the coomassie-stained gel. The purified protein solution was desalted and concentrated using Amicon Ultra-15 (Millipore, 10,000 cut-off) to its final composition [12 mg ml−1 Keap1, 150 mM sodium chloride, 20 mM DTT, 20 mM Tris–HCl pH 8.3] and stored at 193 K.
3
Deglycosylation and Desialylation of UMOD
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The PNGase F (P0704) and α2‐3,6,8,9 Neuraminidase A (P0722) were purchased from New England BioLabs Inc The experiments were carried out according to the manufacturer's instructions with some modifications. N‐glycan was removed both under denatured condition and native condition. Under denatured condition, UMOD was mixed with glycoprotein denaturing buffer, boiled for 10 minutes at 100℃, and cooled down on ice. And then, Glycobuffer 2, NP‐40, and PNGase F were added to the mixture and incubated at 37℃ for overnight. Under native condition, UMOD, GlycoBuffer 2 and PNGase F were directly mixed and incubated at 37℃ for overnight. Sialic acids were removed by α2‐3,6,8,9 Neuraminidase A in a mixture of UMOD and GlycoBuffer 1, and incubated at 37℃ overnight. After incubation, the mixtures were subjected to western blotting to verify the removal of carbohydrates. And then, the mixtures were transferred to 30,000 NMWL filter (AmiconTM Ultra‐15, Milipore) and the buffers were replaced with water by centrifugation at 3000 rpm at 4℃ for 10 times. Then the protein concentration was determined by NanoDrop 1000.
4
Optimized Proteome Profiling
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For proteomic analysis, four biological replicates were analysed per condition. Protein concentration in the sample was quantified with the micro BCA TM protein assay kit (Pierce) following the manufacturer's protocol. After that, 800µg of protein per sample were digested with trypsin following a large-scale filter aided sample preparation (LFASP) method on 10 kDa filters (Amicon TM Ultra-15, Millipore) 16 . Tryptic digests were dried and resuspended in 700µl of IAP buffer (50mM MOPS pH 7.2; 10mM disodium phosphate; 50mM sodium chloride).
5
Efficient Extracellular Vesicle Depletion
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EV-depleted medium was prepared as previously described [12 (link),13 (link),96 (link)]. In summary, FBS was filtered using Amicon® Ultra-15 centrifugal filters with a 100 kDa cut-off (Merk KGaA, Darmstadt, Germany) at 3000× g for 55 min. The EV-depleted FBS was 90% depleted of nanoparticles and used at a 10% concentration to supplement all complete culture media specific to each cell type mentioned above.
6
HDL-mimetic nanoparticles for targeted drug delivery
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The HDL-mimetic peptide-lipid nanoparticles (HPPS) which were modified by DSPE-PEG (2000) maleimide around its surface were prepared and purified as reported before [9 (link),10 (link)]. 27μl Traut’s reagent (25 mg/ml, Sigma Aldrich, USA) was mixed with 360μl anti-TfR mAb (10 mg/ml) in PBS. The mAb-SH could not be collected until 1h reaction of the mixture under a roller mixer at room temperature. Then a Ultrafiltration device (Amicon Ultra-15, 30000 MWCO, MERK) was used to remove the excess Traut’s reagent. The anti-hTfR mAb conjugated HPPS (HPPS-mAb) was prepared by mixing mAb-SH with maleimide-containing HPPS and shaking at room temperature for 20h. The nanostructure carried DIR-BOA, a near-infrared fluorescent dye as cargo. Concentration of DIR-BOA was determined from a standard curve of DIR-BOA according to the same protocol as described before [10 (link),11 (link)].
7
Plasma Sample Preparation for Analyte Quantification
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Filtrate samples were pretreated by protein precipitation with 50% MeOH and acetonitrile. Blank filtrate was prepared using plasma obtained from healthy volunteers. Plasma sample was centrifuged in an Amicon® Ultra‐15 centrifugal filter device (Merk Millipore Ltd.) at 20,600 × g at 4°C until almost all the plasma was filtered, and the supernatant was collected. For calibrator and QC samples, 50 μl of blank filtrate was transferred to a 2‐ml polypropylene tube, and 50 μl of each calibrator or QC sample in 50% MeOH, 25 μl of IS working solution in 50% MeOH, and 100 μl of acetonitrile were added in that order. For patient filtrate and blank filtrate samples, 50 μl of filtrate sample, 25 μl of IS solution in 50% MeOH, 100 μl of acetonitrile, and 50 μl of 50% methanol were added in that order into a polypropylene tube. The mixtures were vortexed for 1 min, and centrifuged at 20,600 × g at 4°C for 10 min. The supernatants were collected and transferred to a 96‐well collection plate (Waters Corp.). The sample plate was sealed with a sealing cap (Waters Corp.) and kept at 4°C until assay. Twenty microliters of sample was injected into the UHPLC.
8
Depletion of EVs from FBS
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The depletion
of EVs in FBS was carried out using a methodology proposed earlier.44 (link) Briefly, FBS was ultrafiltered using Amicon
Ultra-15 centrifugal filter devices (100 kDa cutoff, MerkMillipore,
Darmstadt, Germany) for 30 min at 5000g. The filtrate
was collected and measured for particle concentration using NTA. For
this study, the vesicle-depleted conditioned medium was used to isolate
and purify EVs by SEC, and the EV preparations met the optimal quality
as per the guidelines prescribed by the International Society for
Extracellular Vesicles (ISEV).45 (link)
of EVs in FBS was carried out using a methodology proposed earlier.44 (link) Briefly, FBS was ultrafiltered using Amicon
Ultra-15 centrifugal filter devices (100 kDa cutoff, MerkMillipore,
Darmstadt, Germany) for 30 min at 5000g. The filtrate
was collected and measured for particle concentration using NTA. For
this study, the vesicle-depleted conditioned medium was used to isolate
and purify EVs by SEC, and the EV preparations met the optimal quality
as per the guidelines prescribed by the International Society for
Extracellular Vesicles (ISEV).45 (link)
9
Depletion of EVs from Fetal Bovine Serum
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The depletion of EVs in FBS was carried out using a methodology proposed earlier [33 (link)]. In brief, FBS was ultrafiltered using Amicon Ultra-15 centrifugal filter devices (100 kDa cutoff, MerkMillipore, Darmstadt, Germany) for 30 min at 5000× g. The filtrate was collected and measured for particle concentration using NTA. For this study, the vesicle-depleted conditioned medium was used to isolate and purify EVs by SEC, and the EV preparations met the optimal requirement as per the guidelines prescribed by the International Society for Extracellular Vesicles (ISEV) [34 (link)].
10
Colorimetric Assay for LPMO Activity
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The peroxidase activity of LPMO was detected as described by Breslmayr et al. [47] at room temperature with the following reagent: 128 mM of citrate/phosphate buffer (100 mM final concentration), 100 mM of 2,6-dimethoxyphenol (10 mM final concentration), and 5 mM of hydrogen peroxide (100 µM final concentration). Enzyme concentration in the reaction mixture was 2 µM. Before using AsLPMO10A, it was concentrated using a 10,000 Mw centrifugal filter Amicon®Ultra-15 (Merk Millipore Ltd., Co. Cork, Ireland), and saturated with copper(II)sulfate (the ratio 1:1) for 1 h at 25°C. The absorbance was measured at 469 nm during 300 s. SmLPMO10A (CBP21) was used as the control.
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