Ultrafree mc filter

Manufactured by Merck Group

Sourced in United States, Germany

The Ultrafree-MC filter is a lab equipment product from Merck Group. It is a centrifugal filter device designed for the rapid and efficient separation of samples. The filter functions by utilizing a hydrophilic PTFE membrane to separate liquid from solid components in a sample through centrifugation.

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Lab products found in correlation

Spd m20a, by Shimadzu (8 mentions) Rid 10a, by Shimadzu (8 mentions) Astra 6, by Wyatt Technology (6 mentions) Superdex 200 10 300 gl, by GE Healthcare (4 mentions) Lc 20 prominence hplc system, by Shimadzu (4 mentions) Superdex 200 10 300 gl column, by Cytiva (2 mentions) Prominence lc20, by Wyatt Technology (2 mentions) Mals heleos dawn8 plus qels detector, by Wyatt Technology (2 mentions) Mals heleos dawn8c plus qels detector, by Wyatt Technology (2 mentions) Superdex 200 10 300 gl column, by GE Healthcare (2 mentions) Asys uvm 340 spectrophotometer, by Harvard Bioscience (2 mentions) Femtotips 2, by Eppendorf (1 mentions) Hplc esi ms ms system, by Agilent Technologies (1 mentions) Poroshell 120 ec c18 3, by Agilent Technologies (1 mentions) Agilent 6460 triple quadrupole tandem mass spectrometer, by Agilent Technologies (1 mentions) Phosphatase inhibitor cocktails 1 and 2, by Merck Group (1 mentions) Econo column, by Bio-Rad (1 mentions) P5726, by Merck Group (1 mentions) Eicompak sc 30ds, by Eicom (1 mentions) Superose 6 increase 10 300 gl column, by Cytiva (1 mentions)

27 protocols using ultrafree mc filter

Microinjection of Fluorescent Tracers

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FPT_Low, which was described in the previous report^{34 (link)}, and TR-Dex (D1828, Thermo Fisher), which was used as a cell tracer, were dissolved in an aqueous solution (0.5% w/v) containing 130 mM KCl, 10 mM K₂HPO₄, and 22 mM NaCl. The solution was filtered using an Ultrafree-MC filter (Millipore, Burlington, MA) and microinjected into the cytoplasm using a glass capillary needle (Femtotips II, Eppendorf, Hamburg, Germany). The volume of the injected solution was estimated to be 2 fl.

Chuma S., Kiyosue K., Akiyama T., Kinoshita M., Shimazaki Y., Uchiyama S., Sotoma S., Okabe K, & Harada Y. (2024). Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature. Nature Communications, 15, 3473.

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DNA Extraction, Digestion, and 8-OHdG Analysis

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DNA extraction and digestion was performed according to a previous report [59 (link)]. Samples were homogenized using lysis buffer. The mixture was centrifuged at 10,000× g for 20 s at 4 °C. The deposit was dissolved in 200 μL of enzyme reaction buffer and treated with RNase and protease K. DNA was obtained by washing in 2-propanol and ethanol followed by centrifugation. DNA was dissolved in 20 μM sodium acetate buffer, pH 4.8, and incubated with 4 μL of nuclease P1 (2000 U/mL) at 70 °C for 15 min. Then, 20 μL of 1.0 M Tris-HCl buffer, pH 8.2, was added, and the sample was incubated with 4 μL of alkaline phosphatase (2500 U/mL) at 37 °C for 60 min. The digested DNA samples were acquired after the addition of 20 μL of 3.0 M sodium acetate buffer, pH 5.1 [53 (link)]. The mixture was filtered through a 0.22 μm Ultra free-MC filter (Millipore, Billerica, MA, USA) to remove proteins. Analysis of the DNA base composition was performed using an HPLC/ESI/MS/MS system (Agilent, Santa Clara, CA, USA) and an Agilent Poroshell 120 EC-C18 3.0 (50 mm × 2.7 μm). Mass spectrometric detection was performed using an Agilent 6460 triple quadrupole tandem mass spectrometer (Agilent, Palo Alto, CA, USA) coupled with an electrospray ionization source. The formation of 8-OHdG was calculated as 8-OHdG/10⁵ dG [22 (link)].

Zhu L., Yu T., Qi X., Gao J., Huang K., He X., Luo H, & Xu W. (2016). Limited Link between Oxidative Stress and Ochratoxin A—Induced Renal Injury in an Acute Toxicity Rat Model. Toxins, 8(12), 373.

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MHC-Peptide Complex Extraction Protocol

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To prepare cells for extraction of MHC–peptide complexes, growing cultures were harvested, and spent medium was removed by centrifugation. Cells were washed twice in cold PBS, and dry cell pellets were snap frozen on dry ice and stored at −80 °C for subsequent lysis. Cells (total number: 6.5e8 of 1102mel, 1.4e8 of 2048mel, 3.4e8 of 1363mel, and 9e8 of 2048EBV) were lysed in a solution of 20 mM Tris HCl, pH 8.0; 150 mM NaCl with 1% CHAPS; 1 mM PMSF; 5 g/mL aprotinin; 10 g/mL leupeptin; 10 g/mL pepstatin A; and 1:100 dilutions of phosphatase inhibitor cocktails I and II (Sigma Aldrich), to prevent potential dephosphorylation of peptides during extraction. The lysate was centrifugated and then run over an Econocolumn (Bio-Rad) containing the pan-HLA-DR-specific mAb L243 bound to recombinant protein. After over-night incubation at 4 °C, peptides were eluted from HLA-DR molecules with 10% acetic acid (HOAc) and separated using a 10-kDa cutoff ULTRAFREE-MC filter (Millipore). Extracts were stored at −80 °C.^{16 (link)}

Malaker S.A., Ferracane M.J., Depontieu F.R., Zarling A.L., Shabanowitz J., Bai D.L., Topalian S.L., Engelhard V.H, & Hunt D.F. (2016). Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma. Journal of proteome research, 16(1), 228-237.

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Concentrating and Preserving Cell Secretions

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Half of the supernatant material from deciliated, serum-starved, and serum-stimulated cells was concentrated using a 250 ml 0.2 μm PES filter unit with low-power house vacuum to reduce the volume (20 or 40 mL from one or two 15-cm dishes, respectively) to 2 mL, and finally a Millipore Ultrafree-MC filter (PVDF 0.2 μm size #UFC30GV100) to reduce the volume to approximately 0.5 mL. 5 μl of concentrated supernatant was pipetted onto an acid-treated glass slide. A 22-mm acid-treated circular glass coverslip was placed on the sample, and the slide was immediately plunged into liquid nitrogen for approximately 5 seconds. After removing the slide, the coverslip was removed and fixed in −20°C 100% methanol for 5 minutes. Alternatively, 400 μm of the sample were loaded into an 8-well glass-bottomed LabTek dish (Sigma-Aldrich, # Z734853), and centrifuged at 3,500 x g for 20 minutes, 4°C. Immunofluorescence staining was performed as described above.

Mirvis M., Siemers K.A., Nelson W.J, & Stearns T.P. (2019). Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding. PLoS Biology, 17(7), e3000381.

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Cytosolic Fraction Extraction from Cells

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Cell pellets were suspended gently in 9 packed cell volumes of homogenization buffer A1 (10 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 25 mM KCl, 0.25 M sucrose) with phosphatase and protease inhibitors added at 1:200 just before use (Sigma Aldrich: P5726, P8340). The suspension was incubated for 10 min on ice to promote cell swelling after which the cells were ruptured using a Dounce homogenizer using 9 strokes with an “A” pestle. The suspension was centrifuged at 12,000 × g for 30 min at 4 °C to remove the mitochondria. The supernatant (cytosolic fraction) was subjected to a second centrifugation at 12,000 × g for 30 min at 4 °C. The final supernatant was filtered through a 0.2 μm Ultrafree MC filter (Millipore) by centrifuging at 12,000 × g for 2 min at 4 °C. Aliquots were flash frozen in liquid nitrogen.

Qaiser F., Trembley J.H., Kren B.T., Wu J.J., Naveed A.K, & Ahmed K. (2014). Protein kinase CK2 inhibition induces cell death via early impact on mitochondrial function*. Journal of cellular biochemistry, 115(12), 2103-2115.

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MALS Analysis of POPG:DHPC Bicelles

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A high-performance liquid chromatography pump (Waters 515) equipped with a 2 mL manual injector setup was connected in-line to DAWN Heleos-II and refractive index detectors (Wyatt Technology Inc., Santa Barbara, CA). The running buffer (10 mM potassium phosphate, pH 6.8) was prepared in ultrapure water, filtered, and degassed. A q = 4 POPG:DHPC bicelle solution was prepared in the above buffer and filtered through an Ultrafree-MC filter (0.22 μm, Millipore) prior to injection of a 2 mL sample (0.063 mM) at a flow rate of 0.3 mL/min (25 °C). Raw light scattering data corresponding to 10 angles at the plateau of the injection (mean of ~100 data points over ~0.5 mL for each angle) were extracted from the software Astra (provided with the instrument) for further analysis and fitting. The errors of the fitted parameters in the analysis of the MALS data were determined from 200 Monte Carlo simulations.

Ceccon A., Kubatova N., Louis J.M., Clore G.M, & Tugarinov V. (2022). Global Dynamics of a Protein on the Surface of Anisotropic Lipid Nanoparticles Derived from Relaxation-Based NMR Spectroscopy. The journal of physical chemistry. B, 126(30), 5646-5654.

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Circadian Regulation of Neurotransmitters in Drosophila

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HPLC was used to quantify dopamine and serotonin levels in fly head extracts. Three- to 5-day-old male flies entrained in a 12:12 LD cycle were frozen by immersion in liquid nitrogen at ZT6 and ZT18 after at least 24-hour recovery from CO₂. Samples were acquired without disturbing the LD cycle. Fly heads were detached from their bodies by gentle vortexing. Per sample, three heads were transferred to an Eppendorf tube containing 100 μl of ice-chilled 0.1 M perchloric acid (pH 3.0) and homogenized using a hand tissue grinder. Homogenates were kept on ice for 10 min to denaturalize the proteins, followed by centrifugation at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to an Ultrafree-MC filter with a pore size of 0.45 μm (Merck Millipore), filtered by centrifugation for 1 hour at 14,000 rpm at 4°C, and stored at −80°C until HPLC analysis. Ten microliters of the filtrate was analyzed using HPLC with electrochemical detection (EICOMPAK SC-30DS, EICOM) using a mobile phase consisting of 80% citrate-acetate buffer (0.1 M) (pH 3.5), methanol (20%) with sodium octane sulfonate (220 mg/liter) and EDTA-2Na (5 mg/liter), pumped at a rate of 360 μl/min through a 3.0 mm by 100 mm column (EICOMPAK SC-30DS, EICOM), as described by the manufacturer.

Coll-Tané M., Gong N.N., Belfer S.J., van Renssen L.V., Kurtz-Nelson E.C., Szuperak M., Eidhof I., van Reijmersdal B., Terwindt I., Durkin J., Verheij M.M., Kim C.N., Hudac C.M., Nowakowski T.J., Bernier R.A., Pillen S., Earl R.K., Eichler E.E., Kleefstra T., Kayser M.S, & Schenck A. (2021). The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects. Science Advances, 7(23), eabe2626.

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Determination of XylT Activity with Xyl5-AA Acceptors

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The assay of XylT activity with Xyl₅-AA acceptors was performed in a reaction mixture containing 50 μL microsomal membranes, 1% Triton-100, 0.1 mM cold uridine diphosphate UDP–Xyl, and 0.01 mM Xyl₅–AA. After incubation at room temperature for about 6 h, the reaction was terminated with 1 μL acetic acid and 1 μL 0.5 M EDTA and centrifuged at 13,040× g for 1 min. The supernatant was filtered through a 0.22 μm Ultrafree-MC filter (Merck Millipore, Billerica, MA, U.S.) and centrifuged at 9,060× g for 1 min. The products were analyzed by reversed-phase (RP) chromatography on Thermo U3000 Series HPLC systems and a Fluorescence Detector-3000 (Ex_320nm, Em_420nm). The Xyl₅–AA products were separated on a 4.6 mm × 150 mm, 5 μm Aglient Eclipse XDB-C18 RP column at a flow rate of 0.5 mL min⁻¹ and a column temperature of 30 °C using a gradient program set to 0 to 2 min (8% B), 2 to 20 min (8% to 20% gradient B). Mobile phase A was 50 mM sodium acetate buffer (pH 4.3), and mobile phase B was acetonitrile.

Li C., Xuan L., He Y., Wang J., Zhang H., Ying Y., Wu A., Bacic A., Zeng W, & Song L. (2018). Molecular Mechanism of Xylogenesis in Moso Bamboo (Phyllostachys edulis) Shoots during Cold Storage. Polymers, 11(1), 38.

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Determining Protein Homogeneity and Mass

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Size-exclusion chromatography combined with multi-angle light scattering was performed to determine the homogeneity and the native molecular mass of all proteins under study. Analyses were performed on an LC20 Prominence HPLC equipped with a refractive index detector RID-10A and the photodiode array detector SPD-M20A (all from Shimadzu, Japan). In-line MALS was analyzed either with a miniDAWN TREOS II MALS (for analysis of Spike) or a Heleos Dawn8+ plus QELS apparatus (Wyatt Technology, United States). Prior to analysis, all proteins were centrifuged (16,000 g, 10 min, 20 °C) and filtered (0.1 µm Ultrafree-MC filter, Merck Millipore). Proper performance of the MALS detectors was validated with bovine serum albumin. Purified Spike was analyzed by injection of a total of 50 µg onto a Superose 6 Increase 10/300 GL column (Cytiva) at a flow rate of 0.25 mL min^–1. The mobile-phase buffer used was PBS supplemented with 10% glycerol (pH 7.4). All other proteins were analyzed by using a Superdex 200 10/300 GL column (Cytiva) equilibrated with PBS plus 200 mM NaCl (pH 7.4). A total of 25 µg of each protein was injected and experiments were performed at a flow rate of 0.75 mL min^–1. Data were analyzed using the ASTRA six software (Wyatt Technology).

Capraz T., Kienzl N.F., Laurent E., Perthold J.W., Föderl-Höbenreich E., Grünwald-Gruber C., Maresch D., Monteil V., Niederhöfer J., Wirnsberger G., Mirazimi A., Zatloukal K., Mach L., Penninger J.M., Oostenbrink C, & Stadlmann J. (2021). Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor. eLife, 10, e73641.

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Protein Oligomeric State Analysis

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The homogeneity
and oligomeric state of the purified proteins were determined by HPLC
and size exclusion chromatography (SEC) coupled to multiangle light
scattering (MALS). A LC20 prominence high-pressure liquid chromatography
(HPLC) system with the refractive index detector RIF-10A, the photodiode
array detector SPD-M20A (Shimadzu), and MALS Heleos Dawn8+ with QELY
detector (Wyatt Technology) was used for analysis. Superdex 200 10/300
GL (GE Healthcare) was equilibrated with running buffer (phosphate
buffered saline (PBS) with 200 mM NaCl (pH 7.4)). Experiments were
performed at a flow rate of 0.75 mL min^–1 and 25
°C, and the resulting data were analyzed using the ASTRA 6 software
(Wyatt Technology). Proper performance of the molar mass calculation
was verified by analysis of a bovine serum albumin sample. A 80 μg
portion of the protein (in 5–100 μL) was loaded per run.
For runs with more than one protein, 80 μg per protein was loaded.
All samples were prepared in running buffer. The samples were further
centrifuged (17000g, 10 min) and filtered through
an Ultrafree-MC filter with a pore size of 0.1 μM (Merck Millipore)
before loading to the column.

Michlits H., Lier B., Pfanzagl V., Djinović-Carugo K., Furtmüller P.G., Oostenbrink C., Obinger C, & Hofbauer S. (2020). Actinobacterial Coproheme Decarboxylases Use Histidine as a Distal Base to Promote Compound I Formation. ACS Catalysis, 10(10), 5405-5418.

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