Amicon ultra 0.5 ml 10k centrifugal filters

Manufactured by Merck Group

The Amicon Ultra-0.5 mL 10K centrifugal filters are a laboratory device used for sample concentration and buffer exchange. The filters have a molecular weight cutoff of 10 kDa and a maximum sample volume of 0.5 mL. The device operates through centrifugation to separate molecules based on their size.

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Lab products found in correlation

Phosphodiesterase 1, by Merck Group (1 mentions) Nuclease p1, by Merck Group (1 mentions) Alkaline phosphatase, by Merck Group (1 mentions) Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions) Flag tag (flag), by Merck Group (1 mentions) E coli bl21 de3 plyss cells, by Merck Group (1 mentions)

Close spelling variants of this product

Amicon Ultra 0.5 10K Centrifugal Filters Amicon Ultra-0.5 mL centrifugal filters Amicon Ultra 0.5 mL 10k Amicon Ultra Centrifugal Filters 10K Amicon Ultra 10K Centrifugal Filters Amicon Ultra-0.5 centrifugal filter Amicon® Ultra-0.5 mL centrifugal Amicon Ultra 0.5 ml filters Amicon 10k centrifugal filter Amicon Ultra 10K filter

5 protocols using amicon ultra 0.5 ml 10k centrifugal filters

Quantitative Nucleoside Analysis of Oxidative DNA Damage

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Control and oxidized gDNA samples were digested into nucleosides by 2 U of Nuclease P1 (Sigma-Aldrich) and 10 nM deaminase inhibitor erythro-9-amino-β-hexyl-α-methyl-9H-purine-9-ethanol hydrochloride (Sigma-Aldrich) at 37 °C for overnight and then 6 U of alkaline phosphatase (Sigma-Aldrich) and 0.5 U of phosphodiesterase I (Sigma-Aldrich) at 37 °C for 3 h. After filtering with Amicon Ultra-0.5 mL 10K centrifugal filters (Merck Millipore), the digested samples were subjected to a ZORBAX Eclipse Plus C18 column (2.1 × 150 mm², 1.8-μm, Agilent). HPLC–MS/MS analysis was carried out with 1290 Infinity LC Systems (Agilent) coupled with a 6495B Triple Quadrupole Mass Spectrometer (Agilent). Detailed HPLC-MS/MS program could be found in previous publication^{15 (link)}.

Liu Y., Hu Z., Cheng J., Siejka-Zielińska P., Chen J., Inoue M., Ahmed A.A, & Song C.X. (2021). Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution. Nature Communications, 12, 618.

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Isolation and Characterization of HSP90 ATPase Activity

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AHA1-FLAG, HA-HSP90 and CRBN-Strep-Strep-FLAG were transiently expressed in HEK293T cells as described above. Proteins were immunoprecipitated using FLAG-, HA-agarose (both Sigma) or Streptactin Superflow Sepharose (IBA Lifesciences) respectively and washed five times with 250mM lysis buffer. Beads were then equilibrated into ATPase buffer (20mM KCl, 20mM MgCl₂, 50mM TrisHCl pH 7.5). Proteins were then eluted using the corresponding FLAG/HA peptides or Desthiobiotin containing Buffer E. Proteins were concentrated and separated from peptides using Amicon Ultra-0.5 mL 10k Centrifugal Filters (Merck Millipore). Proteins were visualized by SDS-PAGE and Coomassie Brilliant Blue staining. The ATPase activity of human HSP90 was then measured using the purified proteins in the Molecular Probes PiPer Phosphate-Assay-Kit (Thermo Fisher) according to the manufacturer’s instructions.

Heider M., Eichner R., Stroh J., Morath V., Kuisl A., Zecha J., Lawatscheck J., Baek K., Garz A.K., Rudelius M., Deuschle F.C., Keller U., Lemeer S., Verbeek M., Götze K.S., Skerra A., Weber W.A., Buchner J., Schulman B.A., Kuster B., Fernández-Sáiz V, & Bassermann F. (2021). The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma. Molecular Cell, 81(6), 1170-1186.e10.

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Recombinant Stm-l Protein Purification and Labeling

Cited 2 times

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A recombinant, nontagged Stm-l
protein was obtained as previously described.^{29 (link)} Briefly, Stm-l was overexpressed in BL21(DE3)pLysS E. coli cells (Merck KGaA, Darmstadt, Germany) and
purified using fractionation with ammonium sulfate, size-exclusion
chromatography, and anion exchange chromatography. Purified protein
was desalted to buffer A and stored at −80 °C. Prior to
all experiments, buffer A was changed to deionized water using Amicon
Ultra-0.5 mL 10 K centrifugal filters (Merck). The protein concentration
was determined spectrophotometrically at 280 nm.
Protein labeling
using Alexa Fluor 488 NHS ester dye (AF488, Thermo Fisher Scientific,
Waltham, Massachusetts, USA; dissolved in DMSO) was performed as in
a previous study.^{31 (link)} The labeled protein
(Stm-l/AF488) concentration and the degree of labeling (DOL) were
determined spectrophotometrically by measuring absorbance at 280 and
494 nm. The calculated DOL was 1.9.

Różycka M.O., Bielak K., Ptak M., Jost B., Melo Rodriguez G., Schoelkopf J., Stolarski J., Dobryszycki P, & Ożyhar A. (2023). Effect of Gel Exposition on Calcium and Carbonate Ions Determines the Stm-l Effect on the Crystal Morphology of Calcium Carbonate. Biomacromolecules, 24(9), 4042-4050.

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Protein Labeling with Bodipy FL

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Purified proteins sLif, sLif_Y99A, MD1 and MD1_Y99A were transferred to 50 mM sodium phosphate buffer (pH 7.4) and concentration was adjusted to 70 μM. To label amino groups, Bodipy FL NHS ester (BDP FL; Lumiprobe), was dissolved in DMSO and added to the protein in 1:10 molar ratio to ensure labeling of single dye per protein molecule (obtained degree of labeling approximately 5%). Free dye was removed after overnight incubation at 4 °C by buffer exchange with Amicon Ultra-0.5 mL 10 K centrifugal filters (Merck-Milipore).

Viegas A., Dollinger P., Verma N., Kubiak J., Viennet T., Seidel C.A., Gohlke H., Etzkorn M., Kovacic F, & Jaeger K.E. (2020). Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation. Scientific Reports, 10, 3578.

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Covalent Modification Assay of GCase

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JZ-5029 in DMSO (0.89 mM, 5 µL, 2 equiv) was added to wild-type GCase (22 µM, 95 µL, 1 equiv) in 0.1 M phosphate buffer (pH 7.0) with 1 mM taurocholate in one portion, immediately vortexed for 5 s, and incubated on ice. At indicated time points, the reaction solution (2 µL) was sampled and diluted (1:3125 dilution) into the assay buffer (50 mM citric acid, 176 mM K₂HPO₄, and 0.01% Tween-20 (v/v) at pH 5.9). After 4 h, the reaction solution was dialyzed by repeated (4×) buffer exchanges with 0.1 M phosphate buffer (pH 7.0) using Amicon Ultra 0.5 mL 10K centrifugal filters (Millipore). The enzyme was adjusted to 22 µM and aliquoted for activity. The diluted solutions were assayed with 4MU-β-Glc using the method detailed above.
A competition covalent modification assay with JZ-4109 was conducted using a method similar to the one described above. Wildtype GCase (22 µM) was pretreated with JZ-4109 (5.5, 11, and 22 µM) and with the same volume of DMSO as a blank control, and then treated with JZ-5029 (44 µM). At the indicated time points, the modification rate by JZ-5029 was monitored by enzyme activity assay.

Zheng J., Chen L., Skinner O.S., Ysselstein D., Remis J., Lansbury P., Skerlj R., Mrosek M., Heunisch U., Krapp S., Charrow J., Schwake M., Kelleher N.L., Silverman R.B, & Krainc D. (2018). β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site. Journal of the American Chemical Society, 140(18), 5914-5924.

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