Amicon ultra

Manufactured by Merck Group

Sourced in United States, Germany, Ireland, United Kingdom, France, Hungary, Japan, Sao Tome and Principe, Morocco, Poland, Canada, Italy

The Amicon Ultra is a centrifugal filter device used for the concentration and purification of macromolecules such as proteins, peptides, and nucleic acids. It operates by applying centrifugal force to separate the sample components based on their molecular weight.

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Lab products found in correlation

Hitrap, by Cytiva (13 mentions) Nanodrop, by Thermo Fisher Scientific (8 mentions) Whatman, by Cytiva (8 mentions) Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Superdex 200, by GE Healthcare (6 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions) Ni nta agarose, by Qiagen (5 mentions) Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) Microloop, by MiTeGen (4 mentions) Solarus 950 plasma system, by Ametek (4 mentions) Tla 120.1 rotor, by Beckman Coulter (3 mentions) Mosquito lcp robot, by SPT Labtech (3 mentions) Laminex plates, by Molecular Dimensions (3 mentions) Vitrobot, by Thermo Fisher Scientific (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Superose 6 column, by GE Healthcare (3 mentions) Osmometer model 3250, by Advanced Instruments (3 mentions) Sequencing grade modified trypsin, by Promega (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Centrifugal filter, by Merck Group (3 mentions)

Spelling variants of this product

Amicon® Ultra-15 3K (11 citations) Amicon ultra filtration columns (11 citations) Amicon Ultra 100 kDa MWCO filters (6 citations) Amicon ultra-spin column (5 citations) Amicon Ultra-0.5 30 kDa (3 citations) Amicon Ultra-0.5 Centrifugal Unit with Ultracel 10 membrane (3 citations) Amicon Ultra-15 centrifuge column (2 citations) Amicon Ultra-0.5 30K Centrifugal Filter Devices (2 citations) Amicon 30K Ultra-0.5 mL Centrifugal Filters (2 citations) Amicon Ultra 100 K MWCO (2 citations)

740 protocols using amicon ultra

Isolation and Characterization of Cell-Derived Secretory Granules

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For isolation of T-cell–derived secreted granules, 2×10⁷ CD8⁺ T cells were isolated from spleen and plated in 10 cm plate precoated with anti-CD3 and anti-CD28 antibodies and high-dose IL-2 (1,000 IU/mL) for 48 hours in T cell media containing 10% FBS pre-centrifuged at 140,000 rpm for one hour to deplete bovine-derived exosomes. Supernatants were collected and concentrated using Amicon Ultra (Merk) with 100kD filter. For isolation of NK cells-derived secreted granules, splenic 3×10⁶ NK cells isolated from naïve mice were plated in 12-well plate incubated with high-dose IL-2 for 48 hr. Supernatants were collected and concentrated using Amicon Ultra (Merk) with 100kD filter. Secreted granules were added to B16F10 cultures for 24–48 hr incubation and the numbers of cell-in-cell structures out of total 200 cells were counted under fluorescent microscope.
For isolation of secreted melanosomes from B16F10, Melan A, 8 × 10⁶ cell were cultured in 10 cm tissue culture plates and let to adhere overnight. Cells were incubated 48 hr in DMEM supplemented with 10% exosome-free FBS. Supernatants was collected and centrifuged for 1 hr on glucose gradient, as previously described (Santana-Magal et al., 2020 (link)).

Gutwillig A., Santana-Magal N., Farhat-Younis L., Rasoulouniriana D., Madi A., Luxenburg C., Cohen J., Padmanabhan K., Shomron N., Shapira G., Gleiberman A., Parikh R., Levy C., Feinmesser M., Hershkovitz D., Zemser-Werner V., Zlotnik O., Kroon S., Hardt W.D., Debets R., Reticker-Flynn N.E., Rider P, & Carmi Y. (2022). Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy. eLife, 11, e80315.

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TWEAK-binding IgGs Purification

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IgGs from serum of one TWEAK-binding antibody positive PsoA patient were purified on Affi-Gel Protein A MAPS II columns (Bio-Rad, CA, USA) in accordance with the instructions of the manufacturer. This patient had high titers of TWEAK-binding antibody as detected by immunoblotting experiments and the serum used for purification was obtained at the time of inclusion before etanercept treatment. IgG eluted fraction was concentrated with Amicon Ultra 15 mL (Millipore, Molsheim, France) before being applied to the TWEAK affinity column made from Pierce NHS activated Agarose resin columns (ThermoScientific (Massachusetts, UE) and rh TWEAK. Purified total serum IgG were incubated during 2 h at room temperature on the column and then the TWEAK column was washed with PBS (0,1 M phosphate sodium, 0,15 M NaCl pH 7,2) before bound anti-TWEAK IgG were eluted by using 0.1 M glycine–HCl buffer (pH 2.5) and were directly neutralized with 1 M Tris (pH 9). TWEAK-binding IgG were concentrated with Amicon Ultra 4 mL (Millipore, Molsheim, France) and IgG concentration of the fraction was quantified by immunonephelometry on a Siemens Healthcare analyzer (BN Prospec, Siemens Healthcare, Saint Denis, France). The specificity of the eluted material was further confirmed by its ability to bind rh TWEAK in immunoblotting experiment.

Guis S., Berbis P., Stephan D., Bertin D., Amatore F., Balandraud N., Lesavre N, & Desplat-Jégo S. (2016). TWEAK-binding autoantibodies are generated during psoriatic arthritis and are not influenced by anti-TNF therapy. Journal of Translational Medicine, 14, 185.

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Ligand Fishing for Protein Interactors

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A modified ligand fishing method was performed (Dixon and Edwards 2010). The exudates from roots of 20 plants that secreted for 1 day were dissolved in 5 ml of distilled water. Since we found root exudates could efficiently degrade proteins, inactivation of the proteases in root exudates was performed by boiling them for 30 min to avoid the degradation of KinD in the following steps. Then, the root exudates were filtered by ultrafiltration to collect the molecules that were smaller than 3 kDa for further use. The purified KinD protein was concentrated to 1 mg/ml by Millipore Amicon Ultra, with a cutoff of 10 kDa. The protein and the root exudates were mixed together with a volume ratio of 1:1 (5 ml for each) and were incubated at 25°C for 10 min. Subsequently, the mixture was transferred to a new Millipore Amicon Ultra with a cutoff of 10 kDa and were centrifuged to discard the uncombined root exudates at the bottom. After that, the complex of the ligand and KinD was transferred to another tube. Methanol was added to the complex at a fourfold volume to denature the protein and release the small molecules bound to it. The solution with potential ligands was then injected into GC-MS and LC-MS for isolation and identification.

Liu Y., Feng H., Chen L., Zhang H., Dong X., Xiong Q, & Zhang R. (2020). Root-Secreted Spermine Binds to Bacillus amyloliquefaciens SQR9 Histidine Kinase KinD and Modulates Biofilm Formation. Molecular plant-microbe interactions : MPMI, 33(3).

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Purification of CA_C0359 Protein from E. coli

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Chemically competent E. coli BL21 (DE3) cells (New England Biolabs) were transformed with p359-intein using the manufacturer’s protocol. Cultures of transformed cells were grown to an OD₆₀₀ of 0.6 in 1 l LB Lennox broth supplemented with 50 µg ml⁻¹ ampicillin at 37°C in a shaking incubator and were then transferred to 21°C for 30 min in a shaking incubator. Expression and purification of CA_C0359 protein was performed using previously established protocols (Cantor & Chong, 2001 ▸ ; Chong et al., 1997 ▸ ; IMPACT, New England Biolabs). The molecular mass of the CA_C0359 protein product is 41.7 kDa.
Partially purified protein was concentrated to 5 ml using an Amicon Ultra centrifugal device (MWCO 9K, Millipore) and dialyzed in a MWCO 9K dialysis cassette (Pierce) into 4 l S-75 buffer (150 mM NaCl, 15 mM Tris pH 7.5) for 24 h at 4°C. The dialyzed protein sample was further purified on a Superdex 75 size-exclusion column (GE Healthcare) in S-75 buffer. The fractions containing the CA_C0359 protein, as determined by SDS–PAGE, were concentrated via an Amicon Ultra centrifugal device (MWCO 9K, Millipore).

Germane K.L., Servinsky M.D., Gerlach E.S., Sund C.J, & Hurley M.M. (2015). Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105. Acta Crystallographica. Section F, Structural Biology Communications, 71(Pt 8), 1100-1108.

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Purification of GFP-nanobody-6xHis

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GFP-nanobody-6xHis was transformed into BL21-CodonPlus (DE3)-RIPL cells (Agilent) and protein expression and purification were performed as described above for Rab5B with slight modifications. His-Trap fractions containing GFP-nanobody-6xHis were pooled and concentrated using a 10 kDa MWCO centrifugal filter (Amicon Ultra, Millipore). Concentrated GFP-nanobody-6xHis was applied to Superdex S75 Increase 10/300 GL Column connected to an AKTA FPLC (Cytvia) and run in a buffer containing 25 mM HEPES, pH 7.4; 50 mM KCl; 1 mM MgCl₂; 1 mM DTT. Peak fractions containing pure GFP-nanobody-6xHis were pooled, concentrated and buffer exchanged to the same buffer supplemented with 10% glycerol using a 10 kDa MWCO centrifugal filter (Amicon Ultra, Millipore). Aliquots were snap frozen in LN2 and stored at −80°C. Protein purity was checked on a Sypro (Thermo Fisher Scientific) stained SDS–PAGE gel.

Christensen J.R., Kendrick A.A., Truong J.B., Aguilar-Maldonado A., Adani V., Dzieciatkowska M, & Reck-Peterson S.L. (2021). Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS–Hook–FHIP complexes. eLife, 10, e74538.

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Isolation and Purification of Adult Worm EVs

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Adult worm culture medium from ~1,100 worms was centrifuged once at 200 × g, and the supernatant was concentrated in 3 or 10 kDa filter tubes (Amicon Ultra, Millipore, Merck, Darmstadt, Germany) according to the manufacturer's protocol. E/S in the filters was washed 3 times with PBS and finally concentrated to a volume of 1,870 μl, of which aliquots were stored at -80°C till further use.
Fractions 5-8 with a density 1.21-1.07 g/ml of bottom-up small iodixanol density gradient isolated adult worm EVs (from ~660 worms) were pooled, diluted 1 : 1 with PBS, and transferred into an 0.5 ml 10 kDa centrifugal filter unit (Amicon Ultra, Millipore, Merck) that was precoated with 20 μg trypsin-digested BSA. Iodixanol was removed in 5 centrifugation steps of 10 minutes at 14,000 × g, by which the sample was fully loaded after three steps followed by two additional washing steps with 300-400 μl PBS. The sample was concentrated to 50 μl by a final centrifugation step of 20 minutes. This final sample was collected by brief reverse centrifugation of the filter and directly used for cryo electron microscopy (EM).

Kuipers M.E., Koning R.I., Bos E., Hokke C.H., Smits H.H, & Nolte-‘t Hoen E.N. (2022). Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield. Journal of Immunology Research, 2022, 5473763.

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Purification of Griffithsin Protein from Transgenic N. benthamiana

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Transgenic leaves of N. benthamiana were ground in liquid nitrogen and extracted at 4 °C into lysis buffer (50 mM sodium phosphate, 50 mM ascorbic acid, 10 mM disodium EDTA, and 1 mM PMSF, pH 7.4). The insoluble material was removed by centrifugation at 14,000 rpm and 4 °C for 30 min, and the extract was passed through a 0.45-μm filter and loaded onto a Profinity IMAC column at the rate of 1 ml/min. The column was washed with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 7.4), and the GRFT protein was eluted from the column twice with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 500 mM imidazole, pH 7.4). Fractions containing GRFT were mixed and concentrated by ultrafiltration using spin columns with a 10-kDa molecular weight cut-off (Amicon Ultra, EMD Millipore, Darmstadt, Germany). The purity of the concentrated GRFT was confirmed by SDS-PAGE and Western blot analysis. Additionally, to further assessment of NT-GRFT activity the protein sample was exchange into PBS buffer by ultrafiltration using spin columns with a 10-kDa molecular weight cut-off (Amicon Ultra, EMD Millipore, Darmstadt, Germany) based on manufacturing instruction.

Habibi P., Soccol C.R., O’Keefe B.R., Krumpe L.R., Wilson J., de Macedo L.L., Faheem M., Dos Santos V.O., Prado G.S., Botelho M.A., Lacombe S, & Grossi-de-Sa M.F. (2018). Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana. Process Biochemistry (Barking, London, England), 70, 45-54.

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Reconstitution of H2R/G(s)/Nb35 Complex

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The H₂R/ND/G_s/Nb35-His complex was formed co-translationally. Reactions were supplemented with final concentrations of 15 ng/µL H₂R-Strep template, 60 µM NDs (DOPG) without His-tag, 10 µM purified G_s heterotrimer and 15 µM Nb35-His. Reactions were incubated for 16 h at 30 °C with gentle shaking. Samples were harvested by centrifugation at 18,000 × g and 4 °C for 10 min and subsequently incubated with 1 U/µL apyrase on ice for 90 min. The H₂R/ND/G_s/Nb35-His complex was purified by IMAC at 4 °C. Samples were diluted 1:3 in IMAC buffer A [100 mM NaCl, 20 mM HEPES, pH 7.4] and loaded and reloaded twice on a pre-equilibrated gravity-flow IMAC column. The column was washed with 4 column volumes IMAC buffer A and 4 column volumes IMAC buffer B (IMAC buffer A + 30 mM IMD). Samples were eluted using IMAC elution buffer (IMAC buffer A + 300 mM IMD) and concentrated using Amicon Ultra—0.5 mL units (50 kDa MWCO, Merck Millipore, Darmstadt, Germany). SEC was performed as described above. Complex containing fractions were pooled and concentrated again using Amicon Ultra—0.5 mL units (50 kDa MWCO, Merck Millipore, Darmstadt, Germany).

Köck Z., Schnelle K., Persechino M., Umbach S., Schihada H., Januliene D., Parey K., Pockes S., Kolb P., Dötsch V., Möller A., Hilger D, & Bernhard F. (2024). Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment. Nature Communications, 15, 1831.

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Ultrafiltration for Luciferase Purification

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To discard additional proteins from the luciferase-containing fractions the ultrafiltration procedure was used. First, the active fraction was filtered on a 50 kDa Amicon ® Ultra centrifugal filter unit (Merck Millipore, Germany). BL activity was measured for the concentrated retentate and the permeate. We found that only the permeate was bioluminescent. The bioluminescent permeate was then concentrated on 30 kDa Amicon ® Ultra centrifugal filter unit (Merck Millipore, Germany). The resulting retentate possessed BL activity while the permeate did not.
Thus this concentrated luciferase sample was used for size exclusion chromatography.

Schultz D.T., Kotlobay A.A., Ziganshin R., Bannikov A., Markina N.M., Chepurnyh T.V., Shakhova E.S., Palkina K., Haddock S.H.D., Yampolsky I.V, & Oba Y. (2018). Luciferase of the Japanese syllid polychaete Odontosyllis undecimdonta. Biochemical and biophysical research communications, 502(3).

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Recombinant Archease Protein Purification

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Human Archease isoform 1 (UniProt A8K0B5; note the deviating translation start sites from UniProt Q8IWT0 by twelve additional residues) was expressed and purified as described previously^{10 (link)}. In short, the fusion protein with an N-terminal His₆ tag followed by a thrombin protease cleavage site was expressed in E. coli BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) cells. The clarified lysate was applied to a 5 mL Ni-NTA column (Cytiva). After tag removal (optional) with thrombin (Cytiva), the sample was re-applied onto the Ni-NTA column to remove the affinity tag. The flow-through fraction was concentrated using a centrifugal filter (Amicon Ultra, MWCO 10 kDa, Sigma) and purified by size-exclusion chromatography with buffer containing 25 mM HEPES, pH 7.5, 100 mM NaCl₂, 5% Glycerol, 1 mM TCEP on HiLoad16-600 Superdex 75 pg column (Cytiva). Archease containing peak fractions were concentrated, flash-frozen in liquid nitrogen and stored at −80 °C. We determined a 260/280 nm ratio of 0.56 for the final protein preparation indicating the absence of nucleic acid contaminants (see Source Data).

Gerber J.L., Morales Guzmán S.I., Worf L., Hubbe P., Kopp J, & Peschek J. (2024). Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease. Nature Communications, 15, 2378.

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