Amicon ultra 0.5 centrifugal filter unit

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, Australia

The Amicon Ultra-0.5 Centrifugal Filter Unit is a laboratory device designed for sample concentration and buffer exchange. It utilizes centrifugal force to rapidly concentrate and desalt macromolecules in sample volumes up to 0.5 mL.

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Lab products found in correlation

Nanodrop, by Thermo Fisher Scientific (8 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Amicon ultra 15 centrifugal filter unit, by Merck Group (4 mentions) Zenotoftm 7600 system, by AB Sciex (2 mentions) Acquity uplc beh c18 column, by Waters Corporation (2 mentions) Hen lysozyme, by Merck Group (2 mentions) Fastprep 24 bead beater device mp, by MP Biomedicals (2 mentions) Glycanassure apts kit, by Thermo Fisher Scientific (2 mentions) Pierce protein g spin plate for igg screening, by Thermo Fisher Scientific (2 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions) Quantum prep plasmid miniprep kit, by Bio-Rad (2 mentions) Sanger sequencing on both ends, by Eurofins (2 mentions) Pgem t easy vector system, by Promega (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions) Wizard plus sv dna purification system, by Promega (2 mentions) Nucleospin tissue kit, by Macherey-Nagel (2 mentions) Gotaq polymerase, by Promega (2 mentions) Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions) Western blot luminol reagent, by Santa Cruz Biotechnology (2 mentions)

135 protocols using amicon ultra 0.5 centrifugal filter unit

Lanthanide-Labeled Antibody Conjugation

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Carrier protein and glycerol-free antibodies were labelled with lanthanide isotopes using Maxpar X8 Antibody Labelling Kits (Fluidigm) according to the manufacturer’s instructions. Briefly, X8 polymer was loaded with the lanthanide isotype in L-buffer, and the metal-loaded polymer purified and washed in C-buffer using an Amicon Ultra-0.5 centrifugal filter unit with 3 kDa cutoff (Millipore-Sigma). At the same time, the antibody was reduced with 4 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution in R-buffer, and purified in C-buffer, using an Amicon Ultra-0.5 centrifugal filter unit with 50 kDa cut-off (Millipore-Sigma).
Both the lanthanide-loaded polymer and the partially reduced antibody were mixed and incubated at 37°C for 90 min. Once the incubation was completed, the conjugated antibody was washed several times with W-buffer using an Amicon Ultra-0.5 centrifugal filter unit with 50 kDa cut-off (Millipore-Sigma), and quantified using a NanoDrop spectrophotometer (280 nm). The antibody was finally resuspended in antibody stabilizer PBS supplemented with 0.05% sodium azide at a final concentration of 0.5 mg/mL and stored at 4°C.

Feriotti C., Sá-Pessoa J., Calderón-González R., Gu L., Morris B., Sugisawa R., Insua J.L., Carty M., Dumigan A., Ingram R.J., Kissenpfening A., Bowie A.G, & Bengoechea J.A. (2022). Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity. Cell Reports, 40(6), 111167.

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Quantitative IgG N-glycan profiling

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Hybridoma supernatants
(500 μL) were concentrated using Amicon Ultra-0·5 Centrifugal
Filter Unit (Millipore Sigma, USA). Bulk IgG from five BALB/c mice
and a human plasma sample (Innovative Research, USA) were used as
controls. Total IgG was purified using Pierce Protein G Spin Plate
for IgG Screening (ThermoFisher, USA), and IgGs were further concentrated
using Amicon Ultra-0·5 Centrifugal Filter Unit (Millipore Sigma,
USA). N-Glycans were released using peptide-N-glycosidase F (PNGase F) and labeled with 8-aminopyrene-1,3,6-trisulfonic
acid (APTS) using the GlycanAssure APTS Kit (ThermoFisher, USA), following
the manufacturer’s protocol. Labeled N-glycans
were analyzed using the 3500 Genetic Analyzer capillary electrophoresis
system. The relative abundance of IgG glycan structures was quantified
by calculating the area under the curve of each glycan structure divided
by the total glycans.

Bordoloi D., Xu Z., Ho M., Purwar M., Bhojnagarwala P., Cassel J., Giron L.B., Walker S., Kulkarni A.J., Ruiz E.T., Choi J., Zaidi F.I., Wu Y., Wang S., Patel A., Ramos S., Smith T., Kulp D., Ugen K.E., Srinivasan A., Abdel-Mohsen M., Humeau L., Weiner D.B, & Muthumani K. (2021). Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein. ACS Pharmacology & Translational Science, 4(4), 1349-1361.

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Monosaccharide Analysis in Dough Samples

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Arabinose, galactose, xylose, Glucose, and fructose were analyzed by high performance anion exchange chromatography equipped with a pulse amperometric detection system (HPAEC-PAD). Before analysis, dough samples diluted in water (1:10, w/v) were filtered by an Amicon Ultra-0.5 centrifugal filter unit (Millipore, Billerica, MA, United States) at 12,000 × g for 10 min to get rid of polymeric molecules. Monosaccharides were separated on a CarboPac PA1 column (250 × 4 mm i.d., Dionex, Sunnyvale, CA, United States) and detected using a Waters 2465 pulsed amperometric detector (Waters, United States). The solvents used were 200 mM NaOH and MilliQ water. A gradient elution was maintained at a constant flow rate of 1 ml/min: 0–31 min, 2 mM NaOH; 31–33 min, 200 mM NaOH; and 33–50 min, 2 mM NaOH, with an additional 10 min washing and regeneration steps. The injection volume was 10 μl. Glucose (Merck, Germany), fructose (Merck), xylose (Merck), arabinose (Merck), and galactose (Merck) were used as external standards and 2-deoxy-D-galactose (Sigma-Aldrich, Germany) was used as the internal standard for quantification.

Xie C., Coda R., Chamlagain B., Varmanen P., Piironen V, & Katina K. (2019). Co-fermentation of Propionibacterium freudenreichii and Lactobacillus brevis in Wheat Bran for in situ Production of Vitamin B12. Frontiers in Microbiology, 10, 1541.

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Quantifying ac4C Modification in RNA

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HPLC-MS/MS was conducted as previously described to determine the ac⁴C to C ratio in total RNA and mRNA (42 (link),43 (link)). Briefly, 200–300 ng of total RNA or mRNA was treated with 1 U of nuclease P1 (Sigma-Aldrich, N8630) in 50 μl of buffer containing 100 mM ammonium acetate (pH 5.5; TCI, A2269), 2.5 mM NaCl and 0.25 mM ZnCl₂ for 2 h at 37°C. This was followed by the addition of 3.5 μl of H₂O, 6 μl of 10 × Antarctic phosphatase buffer (NEB, B0289S) and 0.5 μl of Antarctic phosphatase (NEB, M0289S) with an additional incubation at 37°C for 2 h. Following digestion, sample volumes were adjusted to 150 μl using distilled deionized water, and the samples were filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit to remove enzymatic constituents (Millipore, UFC500396). After lyophilization, the samples were reconstituted in 50 μl of distilled deionized water (LC/MS grade) containing 20% acetonitrile (Thermo Fisher Scientific, A955-1), centrifuged three times at 12 000 × g for 15 min and 5 μl of the solution was injected into LC-MS/MS (SCIEX, QTRAP^® 6500⁺ LC-MS/MS, USA).

Chen L., Wang W.J., Liu Q., Wu Y.K., Wu Y.W., Jiang Y., Liao X.Q., Huang F., Li Y., Shen L., Yu C., Zhang S.Y., Yan L.Y., Qiao J., Sha Q.Q, & Fan H.Y. (2022). NAT10-mediated N4-acetylcytidine modification is required for meiosis entry and progression in male germ cells. Nucleic Acids Research, 50(19), 10896-10913.

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In Vitro Transcription/Translation of cPLA2 Variants

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The pDHFR-cPLA2 wt, cPLA2A4T (cPLA2 T268A), cPLA2A4S (cPLA2 S505A), and cPLA2 DM plasmids were used for in vitro transcription/translation using the PURExpress in vitro Protein Synthesis Kit (catalog #E6800S, New England BioLabs). Proteins (wild-type, two single mutants and double mutants) were purified using 50 kDa an Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore). The products were confirmed using SDS-PAGE both before and after purification.

Paul S., Fatihi S., Sharma S., Kutum R., Fields R., Pant H.C., Thukral L, & BK B. (2022). Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson’s Disease. eNeuro, 9(6), ENEURO.0180-22.2022.

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Biotinylated RBD-chAb Binding ELISA

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RBD-chAbs were biotin-labeled using EZ-Link Sulfo-NHS-Lc-Biotin (Thermo; following manufacturer recommendations) and purified using an Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore). Each RBD-chAb (50 ng/well) was pre-coated to ELISA plates. RBD-His or EpEx-His protein (5 ng/well) in bovine serum albumin (BSA) was added to capture Ab-pre-coated ELISA plates, followed by the addition of RBD-chAb (7.8 ng/well) in BSA. Then plates were added biotinylated antibodies (0.78 ng/well) in BSA and incubated at 25°C for 1 h, and 50 μl of 2000-fold diluted Peroxidase Streptavidin (Jackson) was added into each well and incubated for 1 h at 25°C. The BSA without biotinylated antibodies was as a control. The plates were washed with PBST between each step. After a final wash, the plates were developed with TMB, and absorbance was read at 450 nm after the reaction was stopped.

Su S.C., Yang T.J., Yu P.Y., Liang K.H., Chen W.Y., Yang C.W., Lin H.T., Wang M.J., Lu R.M., Tso H.C., Chung M.J., Hsieh T.Y., Chang Y.L., Lin S.C., Hsu F.Y., Ke F.Y., Wu Y.H., Hwang Y.C., Liu I.J., Liang J.J., Liao C.C., Ko H.Y., Sun C.P., Wu P.Y., Jan J.T., Chang Y.C., Lin Y.L., Tao M.H., Hsu S.T, & Wu H.C. (2021). Structure-guided antibody cocktail for prevention and treatment of COVID-19. PLoS Pathogens, 17(10), e1009704.

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Sucrose Fraction Purification Protocol

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One hundred and fifty microliters from each sucrose fraction were transferred to a new 1.5 ml microcentrifuge tube and filled up with washing buffer (20 mM Tris-HCl pH 7.9, 140 mM KCl, 5 mM MgCl₂) to 500 μl. The entire volume was then loaded onto an Amicon Ultra-0.5 Centrifugal Filter Unit (Milipore) and centrifuged at 4°C for 30 min at 14 000 × g. A 7 μl 4× LDS sample buffer (Life Technologies) and 3 μl 10× sample reducing agent (Life Technologies) were added to each sample (17 μl), which was then heated at 70°C for 10 min. Subsequently, 14 μl were loaded on the gel.

Arnold A., Rahman M.M., Lee M.C., Muehlhaeusser S., Katic I., Gaidatzis D., Hess D., Scheckel C., Wright J.E., Stetak A., Boag P.R, & Ciosk R. (2014). Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes. Nucleic Acids Research, 42(21), 13353-13369.

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Fluorescent Labeling of Diverse Proteins

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The His-CRY2 protein was fluorescently labelled using His Lite™ Cy3 Bis NTA-Ni Complex (AAT Bioquest®, 12610). The protein and dye were mixed at a 1:1 molar ratio and incubated at 4 °C for at least 2 h. The TCP22 protein was labelled using iFluor™ 488 succinimidyl ester (AAT Bioquest^®, Cat:1023). The His-PPK1 and His-LWD1 proteins were labelled using iFluor™ 405 succinimidyl ester (AAT Bioquest^®, Cat:1021). The proteins were labelled using the protocol for indicated dye. In brief, the protein was diluted at 1 mg ml⁻¹ in PBS and mixed with 100 mM sodium bicarbonate. The reaction was incubated for 15 min at room temperature and then incubated for 1 h on ice. Fluorescently labelled proteins were purified from the unreacted dye substrate by Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore, UFC500308).

Mo W., Zhang J., Zhang L., Yang Z., Yang L., Yao N., Xiao Y., Li T., Li Y., Zhang G., Bian M., Du X, & Zuo Z. (2022). Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock. Nature Communications, 13, 2631.

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Proteomic Analysis of Brain Fractions

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The mice were transcardially perfused with ice-cold saline. The brains were removed and homogenized in 1 mM Tris/EGTA (pH 7.5) and centrifuged at 13,000 × g for 10 min. The supernatant was collected and used as brain lysate. For digestion experiments, brain lysate was incubated with pronase (1 U/ml, Roche), DNase I (100 μg/ml, Sigma-Aldrich), or RNase (100 μg/ml, Roche) at 37 °C for 1 h. For sucrose-density gradient centrifugation, the brain lysate was ultracentrifuged at 100,000 × g for 1 h. The supernatant was layered on a 10–40% (w/w) linear sucrose gradient in DMEM and centrifuged at 100,000 × g for 16 h. Sucrose was depleted from each sucrose gradient fraction by ultrafiltration using an Amicon Ultra-0.5 centrifugal filter unit (0.5 ml, 3 K, Millipore). We collected fractions 7 and 8, which strongly induced cell proliferation. These fractions were applied to an anionic column (MonoQ, GE Healthcare), and the eluate was condensed by ultrafiltration with an Amicon Ultra-0.5 centrifugal filter unit followed by trypsin treatment and nano-LC-MS/MS, using a Synapt G2 HDMS mass spectrometer (Waters).

Lin H., Muramatsu R., Maedera N., Tsunematsu H., Hamaguchi M., Koyama Y., Kuroda M., Ono K., Sawada M, & Yamashita T. (2017). Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis. EBioMedicine, 27, 71-85.

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Liposomal Drug Encapsulation Protocol

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TPGS was given as a gift from BASF (Ludwigshafen, Germany). Oleic acid, PBS, acetonitrile, and methanol (MeOH) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). LPV and RTV were purchased from AK Scientific (Union City, CA, USA) and USP (Rockville, MD, USA), respectively. The Amicon Ultra-0.5 centrifugal filter unit with a molecular weight cutoff of 100 kDa was purchased from Millipore (Bedford, MA, USA). KH₂PO₄ and NaOH were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Tanaudommongkon I., Tanaudommongkon A, & Dong X. (2021). Development of In Situ Self-Assembly Nanoparticles to Encapsulate Lopinavir and Ritonavir for Long-Acting Subcutaneous Injection. Pharmaceutics, 13(6), 904.

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