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Ultracel 30k

Manufactured by Merck Group
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Ultracel-30K is a laboratory centrifugal filter device designed for the concentration and purification of macromolecules. It features a 30,000 molecular weight cutoff membrane that allows the separation of small molecules from larger biomolecules.

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Lab products found in correlation

Coomassie blue r 250, by Merck Group (1 mentions) Gelatin, by Merck Group (1 mentions) Cary 50, by Agilent Technologies (1 mentions) Pvuii, by New England Biolabs (1 mentions) Amicon ultra 15, by Merck Group (1 mentions) Plasmin, by Roche (1 mentions) Dmem hams f 12 50 50 mix, by Corning (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions)

14 protocols using ultracel 30k

1

Fluorescent Labeling of Recombinant Mononucleosomes

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For in vitro binding studies, commercial recombinant mononucleosomes (rMN), or rMN containing H3K27 modifications (Active Motif) were first end-labelled with aminoallyl dUTP using recombinant Terminal Deoxynucleotidyl Transferase (TdT), and then concentrated by spin-dialysis (Ultracel 30 K, Millipore). The dialyzed rMNs were then labeled with either Alexa Fluor 488 (Green-Fluorescent) or Alexa Fluor 647 (Red-Fluorescent) with succinimidyl ester labeling kits (ARES DNA Labeling Kit, Invitrogen, Cat A21665 and A21676). The fluorescent labelled rMN were concentrated by spin-dialysis.
Zhang S., Postnikov Y., Lobanov A., Furusawa T., Deng T, & Bustin M. (2022). H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors. Communications Biology, 5, 159.
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2

Assembly and Purification of PKA Nanodiscs

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PKA nanodiscs were assembled based on a protocol adapted from previous publications (Nath et al., 2007 (link); Ye et al., 2010 (link)). In brief, DMPC and DMPG were solubilized in chloroform or a chloroform/methanol mixture, mixed thoroughly, and dried onto a glass tube under steady flow of nitrogen. The homogeneous lipid mixture was then solubilized in 100 mM cholate in 10 mM Tris and 100 mM NaCl, pH 7.4, giving a lipid concentration of 50 mM. 72 μl of the lipid solution was then mixed with 200 μl of 200 μM membrane scaffold protein (MSP) in dH2O and 200 μl of 10 μM purified PKA holoenzyme. The final ratio of lipids/MSP/protein is 90:1:0.05 in a total volume of 472 μl. The PKA nanodiscs were assembled by removing the detergents with SM-2 Biobeads overnight at room temperature. The assembled PKA nanodiscs were then purified with a hi-load 16/60 Superdex 200 size exclusion column with 20 mM Tris, 150 mM NaCl, 0.5 mM MgCl2, and 10mM AMPPNP, pH 7.4 for RI and 20 mM MES and 150 mM NaCl, pH 5.8 for RII, as the column buffer. Different buffers were used for RI and RII because of obtaining stable protein. The PKA RII holoenzyme nanodiscs and empty nanodiscs were readily separated and the successful assembly was verified by SDS-PAGE analysis. When necessary, the nanodiscs were further concentrated using Ultracel-30k (Millipore).
Zhang P., Ye F., Bastidas A.C., Kornev A.P., Wu J., Ginsberg M.H, & Taylor S.S. (2015). An Isoform specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes. Structure (London, England : 1993), 23(9), 1563-1572.
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3

Purification of XCAP1-actin Complex

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Extracts from X. laevis oocytes were prepared and applied to an affinity column in which GST-fused Xenopus ADF/cofilin (XAC) had been immobilized as described (35 (link)). Proteins bound to the column were eluted with 1 M NaCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% NaN3, and 20 mM HEPES-KOH, pH 7.2. The eluate was fractionated with ammonium sulfate at 45% saturation. The precipitates obtained by centrifugation were dissolved and dialyzed against 60 mM NaCl buffer (60 mM NaCl, 0.5 mM DTT, 0.01% NaN3, and 20 mM Tris-HCl, pH 8.0), applied to a DE52 column pre-equilibrated with the same buffer, and then eluted with a linear gradient of 60 to 300 mM NaCl. The fractions containing XCAP1–actin complex were directly applied to a hydroxyapatite column pre-equilibrated with 60 mM NaCl buffer and washed thoroughly with the same buffer. XCAP1–actin complex was eluted with a linear gradient of 0 to 300 mM potassium phosphate buffer at pH 8.0. Purified XCAP1–actin complex was concentrated by ultrafiltration with Ultracel-30K (Millipore) and dialyzed against 0.1 M KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NaN3, and 20 mM HEPES-KOH, pH 7.2.
Kodera N., Abe H., Nguyen P.D, & Ono S. (2021). Native cyclase-associated protein and actin from Xenopus laevis oocytes form a unique 4:4 complex with a tripartite structure. The Journal of Biological Chemistry, 296, 100649.
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4

Hydrogel Degradation and Tube Formation

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Hydrogels were formed, stored in buffer at 37 °C fo r 24 hours, and then degraded in 1 mL of 10 nM MMP2 containing buffer. To maintain MMP bioactivity without increasing degradation solution volume, additional MMP2 (10 μmol/mL) was introduced every other day until hydrogels had fully degraded. Degraded hydrogels were collected, the MMP2 removed by centrifugal filtration (Ultracel 30K, Millipore), and the amount of “2T” peptide released determined as previously described (section 2.10). Degraded gel solutions were diluted in control media for the tube formation assay such that the final concentration of released SPARC118(2T) was 100 nM. The degraded gel:media ratio was kept constant across all degraded gel types. Degraded gel bioactivity was then assessed using the tube formation assay (~1/7,000th of a gel per well), as previously described (section 2.4), with control media containing buffer at equivalent amounts as the degraded gel groups.
Van Hove A.H., Beltejar M.J, & Benoit D.S. (2014). Development and in vitro assessment of enzymatically-responsive poly(ethylene glycol) hydrogels for the delivery of therapeutic peptides. Biomaterials, 35(36), 9719-9730.
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5

Gelatin Zymography Assay for BRMS1

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2×106 cells were seeded in 100 mm plate for 24 hour, cells were transfected with the BRMS1 and control plasmids. 24 hours after transfection, serum-free medium was applied to the cells overnight and the proteins in the conditioned medium were concentrated with Ultracel-30k centrifugal filters (Millipore, Billerica, MA) at 5,000×g for 20 min at 4°C. Proteins (50 µg) were loaded on a 10% polyacrylamide gel containing 0.1% gelatin (Sigma). After electrophoresis, gel was incubated in Triton X-100 exchange buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM CaCl2 and 2.5% Triton X-100) for 30 min followed by 10 min wash with incubation buffer (same buffer without Triton X-100) thrice. The gel was then incubated in incubation buffer overnight at 37°C, stained with 0.5% Coomassie blue R250 (Sigma) for 4 hour and destained with 30% methanol and 10% glacial acetic acid for 2 hour. gelatinolytic activity was shown as clear areas in the gel.
Mei P., Bai J., Shi M., Liu Q., Li Z., Fan Y, & Zheng J. (2014). BRMS1 Suppresses Glioma Progression by Regulating Invasion, Migration and Adhesion of Glioma Cells. PLoS ONE, 9(5), e98544.
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6

Hydrogel Degradation and Angiogenesis Assay

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Hydrogels were formed, incubated overnight in buffer at 37 °C, and degraded with 10 nM MMP2 (1 mL/gel). To ensure activity, MMP2 (10 μmol/mL) was supplemented every 48 hours until hydrogels had fully degraded. MMP2 was then removed from the degraded gel solution by centrifugal filtration (Ultracel 30K, Millipore), and the amount of “2T” peptide in solution determined as previously described (section 2.2.3). Degraded gel solutions were diluted in control media and used in the tube formation assay (section 2.3.1), maintaining the buffer: media ratio across all groups.
Van Hove A.H., Burke K., Antonienko E., Brown E I.I.I, & Benoit D.S. (2015). Enzymatically-responsive pro-angiogenic peptide-releasing poly(ethylene glycol) hydrogels promote vascularization in vivo. Journal of controlled release : official journal of the Controlled Release Society, 217, 191-201.
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7

Encapsulation Efficiency of GAN Formulations

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The encapsulation efficiency (EE) of GAN formulations is calculated according to the following equation [56 (link)]:
After ultra-filtration (20 min at 4000× g) using Ultracel-30K Millipore filters, the absorbance of free GA in the supernatant was measured at 270 nm using UV–visible spectroscopy (Cary 50, Varian, Belrose, Australia), and the amount of GA was calculated on the basis of the standard curve.
Zolghadri S., Asad A.G., Farzi F., Ghajarzadeh F., Habibi Z., Rahban M., Zolghadri S, & Stanek A. (2023). Span 60/Cholesterol Niosomal Formulation as a Suitable Vehicle for Gallic Acid Delivery with Potent In Vitro Antibacterial, Antimelanoma, and Anti-Tyrosinase Activity. Pharmaceuticals, 16(12), 1680.
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8

Plasmid Construction for Methylation Quantification

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Plasmid constructs containing bisulfite-converted sequences that represent exactly 0%- (M0) and 100%-methylation (M100) of the INK4A (P14), CDKN2A (P16), and MLH1 genes were synthesized and purified using high-performance liquid chromatography through a commercial service (Bioneer, Daejeon, Korea). The sequence information for the synthesized inserts is presented in S1 Data. Sequence-verified plasmid DNA (3–4 μg) was linearized by PvuII (New England Biolabs) and purified by ultrafiltration (Ultracel-30K; Millipore). Linearized plasmids were used as stock materials for preparation of working standards.
Yu H., Hahn Y, & Yang I. (2015). Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation. PLoS ONE, 10(9), e0137006.
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9

HSA Reduction and Modification Protocol

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Commercial 20% solution of HSA contains a non-physiological, low concentration of reduced HSA-SH (around 20%). For experimental purposes, obtained FA-free HSA was reduced with dithiothreitol as described by Penezić [5 (link)]. Briefly, before the reduction, the content of the HSA-SH group was determined. Then, FA-free HSA in 0.1 M sodium phosphate buffer, pH 7.4, was mixed with dithiothreitol at a molar ratio of 1:1 (molar content of oxidized thiol group:dithiothreitol). After incubation for 1 h at 37 °C, dithiothreitol was washed away from HSA with 0.1 M sodium phosphate, pH 7.4, using an Ultracel-30K device (Millipore, Bedford, MA, USA). After this treatment, the HSA-SH content was 0.89–0.92 mol SH group per mol HSA. For the incubation of HSA with FAs and glucose experiments, HSA-SH content was adjusted to 0.55 mol -SH/mol HSA by mixing appropriate volumes of highly reduced FA-free HSA and FA-free HSA.
Uzelac T., Smiljanić K., Takić M., Šarac I., Oggiano G., Nikolić M, & Jovanović V. (2024). The Thiol Group Reactivity and the Antioxidant Property of Human Serum Albumin Are Controlled by the Joint Action of Fatty Acids and Glucose Binding. International Journal of Molecular Sciences, 25(4), 2335.
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10

Purification of XCAP1-Actin Complex

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Extracts from Xenopus laevis oocytes were prepared and applied to an affinity column in which GST-fused Xenopus ADF/cofilin (XAC) had been immobilized as described (34) . Proteins bound to the column were eluted with 1 M NaCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% NaN3, and 20 mM HEPES-KOH, pH 7.2. The eluate was fractionated with ammonium sulfate at 45% saturation. The precipitates obtained by centrifugation was dissolved and dialyzed against 60 mM NaCl buffer (60 mM NaCl, 0.5 mM DTT, 0.01% NaN3, and 20 mM Tris-HCl, pH 8.0), applied to a DE52 column pre-equilibrated with the same buffer, and then eluted with a linear gradient of 60-300 mM NaCl. The fractions containing XCAP1-actin complex was directly applied to a hydroxyapatite column pre-equilibrated with 60 mM NaCl buffer and washed thoroughly with the same buffer. XCAP1-actin complex was eluted with a linear gradient of 0-300 mM potassium phosphate buffer at pH 8.0. Purified XCAP1-actin complex was concentrated by ultrafiltration with Ultracel-30K (Millipore) and dialyzed against 0.1 M KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NaN3, and 20 mM HEPES-KOH, pH 7.2.
Kodera N., Abe H., & Ono S. (2020). Native cyclase-associated protein and actin fromXenopus laevisoocytes form a 4:4 complex with a tripartite structure.
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