A 4-fold molar excess of the spin label reagent MTSL (1-oxy-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate (Toronto Research Chemicals) dissolved in acetonitrile was added to H3K36me3 peptide (residues 32-41) with a Gly33Cys substitution (SIGMA Genosys), dissolved in buffer [10 mM potassium phosphate (pH 6.8), 20 mM NaCl], and incubated at 25°C for 16 h in the dark. Unreacted MTSL was removed by using a PD MidiTrap G-10 column (GE Healthcare) equilibrated with distilled water. The spin labeled peptide eluted with water was lyophilized and dissolved in buffer, and the pH was adjusted to 6.8. A paramagnetic sample was obtained by adding MTSL-H3K36me3 peptide to 15N-labeled Eaf3 CD dissolved in buffer at a molar ratio of 1:1 (final concentration 0.3 mM), and the 1H,15N-HSQC spectrum was recorded. A diamagnetic sample was generated by reducing the paramagnetic sample with a 3.2-fold molar excess of ascorbate (Wako) at 25°C for 4 h in the dark. After the reduction, the 1H,15N-HSQC spectrum was recorded.
Pd miditrap g 10 column
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
The PD MidiTrap G-10 columns are size-exclusion chromatography columns designed for the rapid desalting and buffer exchange of protein samples. These columns effectively separate molecules based on their size, allowing the removal of small molecules such as salts, while retaining the desired proteins.
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Lab products found in correlation
9 protocols using pd miditrap g 10 column
1
Spin Label Tagging of H3K36me3 Peptide
2
Fluorescein Conjugation of Ovalbumin
3
Preparation of Nuclear Extracts from NB4 Cells
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Nuclear extracts from NB4 cells were prepared as described previously (Dignam et al., 1983 (link)) with slight modifications. NB4 cells were harvested by centrifugation at 1000g, 4 min at 4°C and washed twice with icecold PBS. Packed cell volume (pcv) was estimated and cells were resuspended in 5xpcv of ice-cold hypotonic buffer (10mM HEPES pH 7.5, 10mM NaCl, 3mM MgCl2) supplemented with protease inhibitors. Cells were incubated on ice for 5 min, followed by addition of dodecyl-β-D-maltosid (DDM) to a final concentration of 0.05%. The sample was vortexed briefly and immediately centrifuged for 5 min at 600g, 4°C. The cytosolic fraction was removed and the nuclei were washed with 20xpcv hypotonic buffer (5 min, 600g, 4°C). The supernatant was removed and the nuclei were washed with 20xpcv PBS (5 min, 600g, 4°C). The nuclei were extracted with 2/3xpcv of high salt buffer (20mM HEPES pH 7.5, 400mM NaCl, 1mM EDTA ph 8, 1mM EGTA pH 8, 20% glycerol, 1mM DTT) supplemented with protease inhibitors while shaking on a tubeshaker at 4°C for 20 min at 750 rpm. Nuclear extracts were cleared by centrifugation at 18000g for 20 min at 4°C and the buffer was exchanged to membrane binding buffer (20mM HEPES pH 7.5, 400mM NaCl, 1mM EDTA ph 8, 1mM EGTA pH 8, 25% glycerol, 1mM DTT) by gel filtration with PD MidiTrap G10 columns (GE healthcare) according to instructions of manufacturers.
4
Purification and Characterization of Enoxaparin Fractions
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The IC-derived fractions of enoxaparin were desalted as described previously [29 (link)] with minor modifications. Each fraction was concentrated on a miVac DNA centrifugal concentrator (Genevac Ltd, Suffolk, UK) at 40°C. The concentrated solutions containing NaCl and enoxaparin fractions were desalted using PD MidiTrap G-10 columns (GE Healthcare Life Sciences, Uppsala, Sweden) having desalting capacity of more than 95%. The recovery of each fraction was determined by reanalysing the desalted fractions using IC under the same chromatographic conditions as described above. The concentration of each fraction was calculated using the differences in the peak areas of the desalted fraction and enoxaparin fraction eluted at the same time.
The stock solution (1 mg/mL) of each fraction was prepared in serum-free Ham’s F12K medium. The fractions were tested for their effects on the release of IL-6 and IL-8 from stimulated human pulmonary epithelial cells, as described above. Anticoagulant activity of the fraction responsible for the inhibitory effect of enoxaparin was determined using an anti-factor Xa assay. Its structural characterisation was then carried out using a nuclear magnetic resonance (NMR) technique.
The stock solution (1 mg/mL) of each fraction was prepared in serum-free Ham’s F12K medium. The fractions were tested for their effects on the release of IL-6 and IL-8 from stimulated human pulmonary epithelial cells, as described above. Anticoagulant activity of the fraction responsible for the inhibitory effect of enoxaparin was determined using an anti-factor Xa assay. Its structural characterisation was then carried out using a nuclear magnetic resonance (NMR) technique.
5
Purification of Bacteriocin EntDD14 from E. faecalis
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EntDD14 was purified from the supernatant of E. faecalis 14 WT, ΔddE, ΔddF, and ΔddF-Comp strains. The purification procedure was adapted from Abriouel et al. [44 (link)] as follows. Each strain was grown in 200 mL of GM17 at 37 °C for 24 h. After harvesting of the cultures by centrifugation (10,000× g during 10 min at 4 °C), the obtained cell-free supernatants were incubated at room temperature for 24 h with the CM Sephadex® C-25 resin (GE Healthcare Life Sciences, Issaquah, WA) with shaking at 90 rpm. The mixture was poured into a chromatography column, where the resin was allowed to settle. Then, the resin was washed with 400 mL of distilled water and 200 mL of 0.5 M NaCl. Then, the resin-bound DD14 was eluted with 30 mL of 1.5 M NaCl. The solution containing DD14 was desalted by gel filtration using PD MidiTrap G-10 columns (GE), eluting with milliQ water. The pure EntDD14 was quantified using the BCA assay protein kit (Sigma-Aldrich) and then, it was dried in aliquots by miVac Sample Concentrators (SP Scientific, Gardiner, NY, USA) for its storage. When used, an aliquot of pure DD14 was resuspended in the appropriate volume of MilliQ water to achieve the desired concentration.
6
Fractionation and Characterization of Enoxaparin
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IC separated enoxaparin fractions were collected and desalted as described previously [24 (link)] with minor modifications. Briefly, collected fractions were concentrated on a miVac DNA centrifugal concentrator (Genevac Ltd, Suffolk, UK) at 40°C and subsequently desalted using PD MidiTrap G-10 columns (GE Healthcare Life Sciences, Uppsala, Sweden). The desalted fractions were kept at 4°C until use. The concentration of IC separated fraction of enoxaparin was determined by constructing a calibration curve from the peak areas of LMWH standards against their known concentrations. The recovery of each desalted fractionwas calculated using the differences in the peak areas of the desalted fraction and enoxaparin fraction eluted at the same time. Each desalted fraction was tested for its anticoagulant activity and its inhibitory effect on cytokine release from activated PBMCs.
7
Purification of the Antimicrobial Peptide EntDD14
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EntDD14 was purified from the supernatant of the WT. The purification procedure was adapted from Abriouel et al. [36 (link)] and was performed as described in Pérez-Ramos et al. [26 (link)]. Briefly, a 24 h cell-free supernatant and the CM Sephadex® C-25 resin (GE Healthcare Life Sciences, Chicago, IL, USA) were incubated together for 24 h at room temperature by shaking at 90 rpm. Then, using a chromatography column, the resin was settled and washed, first with distilled water and then with 0.5 M NaCl. The EntDD14 linked to the resin was eluted with 1.5 M NaCl. Afterwards, using PD MidiTrap G-10 columns (GE), the solution of EntDD14 was desalted with MilliQ water. The pure EntDD14 was then quantified with the BCA assay protein kit (Sigma-Aldrich, St. Louis, MO, USA) and dried out with the miVac Sample Concentrator (SP Scientific, Warminster, PA, USA) for storage. When used, an aliquot of pure EntDD14 was resuspended in the appropriate volume of MilliQ water to obtain the desired concentration.
8
Purification of EntDD14 Bacteriocin
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A volume of 200 mL culture of Ent. faecalis 14 was grown for 24 h in GM17 medium at 37°C without shaking. The culture supernatant was harvested by centrifugation (8,000 rpm) and was incubated for 24 h at 4°C with CM Sephadex® C-25 (GE Healthcare Life Sciences, Milwaukee, WI). The resin was then washed with 5 bed volumes (BV) of 50 mM sodium phosphate (pH 6.3) and 5 BV of 0.5 M NaCl. The resin-bound bacteriocin was then eluted with 2 BV of 1.5 M NaCl. The EntDD14 was then further purified by gel filtration using PD MidiTrap G-10 columns (GE Healthcare Life Sciences) in Milli-Q water. The eluted solution was dried by miVac Sample Concentrators (SP Scientific NY-USA) and the dried samples were then resuspended in appropriate volumes of Milli-Q water to give the desired concentration. After each purification step, protein concentration was measured using a bicinchoninic acid assay (Sigma-Aldrich). The purity of the EntDD14 was verified by MALDI-TOF/MS analyses (Bruker Daltonics, Bremen, Germany) according to the recommendations of the manufacturer.
9
Purification and Characterization of EntDD14
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EntDD14 was puri ed from the supernatant of E. faecalis 14 WT, ΔddE, ΔddF and ΔddF-Comp strains. The puri cation procedure was adapted from Abriouel et al. [24] as follows. Each strain was grown in 200 mL of GM17 at 37 °C for 24 h. After harvesting of the cultures by centrifugation (10,000×g during 10 min at 4°C), the obtained cell-free supernatants were incubated at room temperature for 24 h with the CM Sephadex® C-25 resin (GE Healthcare Life Sciences) with shaking at 90 rpm. The mixture was poured into a chromatography column, where the resin was allowed to settle. Then, the resin was washed with 400 mL of distilled water and 200 mL of 0.5 M NaCl. The resin-bound DD14 was then eluted with 30 mL of 1.5 M NaCl. The solution containing DD14 was desalted by gel ltration using PD MidiTrap G-10 columns (GE), eluting with milliQ water. The pure EntDD14 was quanti ed using the BCA assay protein kit (Sigma-Aldrich) and then, was dried in aliquots by miVac Sample Concentrators (SP Scienti c, USA) for its storage. When used, an aliquot of pure DD14 was resuspended in the appropriate volume of MilliQ water to achieve the desired concentration.
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