Amicon ultra 15 10k centrifugal filters

Manufactured by Merck Group

The Amicon Ultra-15 10K Centrifugal Filters are a laboratory device used for the concentration and purification of samples. The filters have a molecular weight cut-off of 10,000 Daltons, allowing for the selective retention of macromolecules while allowing smaller molecules to pass through.

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Lab products found in correlation

Histrap hp column, by GE Healthcare (3 mentions) Hiload 26 600 superdex 200 pg column, by GE Healthcare (2 mentions) Hiload 16 600 superdex 75 pg column, by Cytiva (1 mentions) Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)

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Amicon Ultra centrifugal filters (758 citations) Amicon Ultra-15 centrifugal filters (239 citations) Amicon Ultra-15 10K (30 citations) Amicon Ultra Centrifugal Filters 10K (23 citations) Amicon Ultra 10K Centrifugal Filters (14 citations) Amicon Ultra 10K filter (13 citations) Ultra-15 Centrifugal Filter (9 citations) Amicon Ultra-15 10K filter (3 citations) Amicon Ultra-15 10K centrifugal (2 citations) Amicon 10k centrifugal filter (2 citations)

4 protocols using amicon ultra 15 10k centrifugal filters

Purification and Binding Analysis of SNX9 and β2-HAD

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GST-SNX9 in a pGEX-KG vector and His6x-β2-HAD in a pET vector (gift of Dr. Linton Traub, University of Pittsburgh, Pittsburgh, PA) were expressed in BL21(DE3) and then affinity purified using glutathione Sepharose 4B beads (ABT) and a HisTrap HP column (GE Healthcare). The affinity-purified GST-SNX9 was thrombin-digested to remove GST. The resulting SNX9 and β2-HAD proteins were separately applied to a HiLoad 26/600 Superdex 200 pg column (GE Healthcare). Peak fractions of target proteins were collected and concentrated using Amicon Ultra-15 10K Centrifugal filters (Sigma-Aldrich).
Binding of TAT, Wbox2_mutant, and Wbox2 to SNX9 and β2-HAD was investigated by ITC using a MicroCal iTC₂₀₀ (GE Healthcare Life Sciences). Measurements were performed at 20°C in reaction buffer: 20 mM Hepes, 150 mM NaCl, and 1 mM TCEP, pH 7.4. Protein and peptide concentrations were determined by absorbance at 280 and 205 nm, respectively. The peptides were injected from a syringe in 21 steps up to a molar excess over the protein concentration. Titration curves were executed with NITPIC and fitted with SEDPHAT. GUSSI was further used to output the figures presented.

Chen Z., Mino R.E., Mettlen M., Michaely P., Bhave M., Reed D.K, & Schmid S.L. (2020). Wbox2: A clathrin terminal domain–derived peptide inhibitor of clathrin-mediated endocytosis. The Journal of Cell Biology, 219(9), e201908189.

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SNX9 and AP2 Binding Assay

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GST-SNX9 in a pGEX-KG vector and His6x-β2-hinge+appendage in a pET vector (gifted from Dr. Linton Traub, Univ. of Pittsburgh) were expressed in BL21(DE3) and then affinity-purified using glutathione Sepharose 4B beads (ABT) and HisTrap HP column (GE Healthcare), respectively. The affinity-purified GST-SNX9 was thrombin-digested to remove GST. The resulting SNX9 and His6x-β2-hinge+appendage proteins were separately applied to a HiLoad 26/600 Superdex 200 pg column (GE Healthcare). Peak fractions of target proteins were collected and concentrated using Amicon Ultra-15 10K Centrifugal filters (Sigma-Aldrich).
Binding of Wbox2 to SNX9 and AP2-β2-hinge+appendage domain was investigated by ITC using a MicroCal iTC200 (GE Healthcare Life Sciences). Measurements were performed in 20mM HEPES, 150mM NaCl, 1mM TCEP, pH=7.4 and at 20°C. Protein and peptide concentrations were determined by absorbance at 280nm and 205nm, respectively. The peptides were injected from a syringe in 21 steps up to a molar excess over the protein concentration. Titration curves were executed with NITPIC and fitted with SEDPHAT. GUSSI was further used to output the figures presented.

Chen Z., Mino R.E., Mettlen M., Michaely P., Bhave M., Reed D.K., & Schmid S.L. (2020). Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME.

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Purification and Characterization of Nop1 and Nop56 Complex

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Purified Nop1^83–327 and Nop56^1–166, or mutants thereof, were mixed in a 1:1 ratio and incubated for 15 min at room temperature before further purification steps. All complexes were purified using size-exclusion chromatography (SEC) on an Äkta pure system at room temperature with running buffer D (50 mM sodium phosphate, 100 mM NaCl, 10 mM β-mercaptoethanol, pH 7.0) or running buffer E for crystallization (50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, pH 7.5). A Superdex 200 Increase 10/300 GL column (Cytiva) or, for the preparation of the crystallization sample, a HiLoad 16/600 Superdex 75 pg column (Cytiva) were used. Purity was assessed using SDS gel-electrophoresis. For crystallization, the purified complex was concentrated using Amicon Ultra-15 10K centrifugal filters (Merck).

Höfler S., Lukat P., Blankenfeldt W, & Carlomagno T. (2021). High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain. RNA, 27(4), 496-512.

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Efficient Purification of Recombinant Proteins

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The purification of recombinant proteins was performed by gravity-flow chromatography with precharged Ni Sepharose^™ (GE Healthcare). The coding sequences of mAldO and variants were cloned into the expression vector pET-30a(+) by employing CPEC method, and the resulting recombinant plasmids were electroporated into E. coli BL21(DE3). E. coli BL21 (DE3) cells were grown in LB medium containing 50 mg/l kanamycin, and recombinant protein expressions were induced by adding 0.1 mM IPTG when the cells reached 0.6–0.8 of OD600nm and cultured at 25°C for 18 h. Cells were harvested and resuspended in 50 mM Tris-HCl buffer (pH 7.5), then disrupted by sonication on ice and supernatants of the cell lysate collected by centrifugation (14 972 g, 30 min, 4°C) (Sigma 3–18 K). The supernatant was filtered through a 0.22 μm filter and loaded onto a pre-equilibrated HisTrap HP column (GE Healthcare). The recombinant protein was eluted with 200 mM imidazole in 20 mM sodium phosphate (pH 7.5) with 0.5 M NaCl. Further protein purification and concentration was obtained by Amicon^® Ultra-15 10 K Centrifugal Filters (MERCK) with 50 mM Tris-HCl (pH 7.5). The purified protein was verified by a 10% SDS-PAGE gel.

Zhang C., Chen Q., Fan F., Tang J., Zhan T., Wang H, & Zhang X. (2021). Directed evolution of alditol oxidase for the production of optically pure D-glycerate from glycerol in the engineered Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 48(7-8), kuab041.

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