Five Haloarchaea strains were cultivated in 250-mL flasks containing 80 mL DSMZ medium 589 with 20 g L−1 sucrose for 5 days (37 °C and 200 r.p.m.). One milliliter culture broth of each strain was extracted from Diaion® HP-20 resin (Sigma-Aldrich), washed three times with 1 mL of H2O, and then collected crude extracts with elution of 1 mL MeOH. After being dried by a vacuum concentrator, all crude extracts were dissolved in methanol and subjected to MALDI-TOF and HR-LCMS.
Diaion hp 20 resin
Manufactured by Merck Group
Sourced in United States
Diaion HP-20 is a non-ionic, macroporous, styrene-divinylbenzene copolymer resin. It is used for adsorption and separation of various organic compounds in liquid chromatography applications.
Automatically generated - may contain errors
Lab products found in correlation
Sephadex lh 20, by Merck Group (2 mentions)
Sephadex lh 20, by GE Healthcare (2 mentions)
Millipore water, by Merck Group (2 mentions)
Zorbax extend c18, by Agilent Technologies (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Autopol 4 automatic polarimeter, by Rudolph Research Analytical (1 mentions)
Apollo c18 column, by Agilent Technologies (1 mentions)
Semipreparative hplc system, by Agilent Technologies (1 mentions)
Spectrum one ftir spectrometer, by PerkinElmer (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rotary evaporator, by Eyela (1 mentions)
Jms 700 mstation, by JEOL (1 mentions)
Fourier transform nmr spectrometer, by Bruker (1 mentions)
D glucosamine hcl, by Merck Group (1 mentions)
Fmoc cl, by Merck Group (1 mentions)
Glycylsarcosine glysar, by Merck Group (1 mentions)
Amantadine hcl, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Amberlite xad 16 resin, by Merck Group (1 mentions)
Tryptic soy agar, by HiMedia (1 mentions)
19 protocols using diaion hp 20 resin
1
Haloarchaeal Metabolite Extraction and Analysis
2
Extraction and Purification of Balloon Flower Leaf
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Balloon flower leaf extract was prepared as previously reported. (Shin et al., 2019 ). A two-year-old balloon flower leaf harvested in Bonghwa-Gun was used to prepare the extract. The balloon flower leaf was dried for 72 h at 40 °C, and then ground using an electric grinder. A crude powder (100 g) as a particle size of about 300–700 μm was extracted and added to 1 L of 80% (v/v) methanol at 70 °C for 24 h without shaking. The methanol extract was filtered, the methanol was completely removed using a rotary evaporator, and the residue was dissolved in the same volume of distilled water. To prevent inhibition of the reactions, the sugars in the extract were removed using a Diaion HP-20 resin (Sigma-Aldrich, St. Louis, MO, USA) column. After loading 1 L of the extract onto the column, the resin that was adsorbed with platycosides was washed with 2 L of distilled water. The column was then eluted with 2 L of methanol at a flow rate of 0.5 mL/min. The methanol in the eluent was evaporated, and 1 L of distilled water was added to the residue. The resulting balloon flower leaf extract was used for the production of deglucosylated platycodin D.
3
Purification of Streptomyces Metabolites
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Streptomyces strains were grown as described above. The culture supernatant was treated with 5% (w/v) Diaion HP-20 resin (Sigma-Aldrich), followed by elution with 10% methanol and 50% ACN using column chromatography. The 50% ACN fraction was applied to a Sephadex LH20 (Sigma-Aldrich) column using 50% ACN as the running solvent. Active fractions were collected and further purified by HPLC on an Agilent semi-prep column (Zorbax Extend C-18, 21.2 × 100 mm, 5 μm) using a linear gradient from 5 to 10% solvent B (solvent A, 0.1% v/v formic acid in water and solvent B, 0.1% v/v formic acid in ACN) over 10 mins to yield pure compound.
4
Analytical Techniques for Natural Product Characterization
5
Extraction of Bread Compounds
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The bread sample was mixed with 10% diaion HP 20 resin (Sigma) under shaking for 30 min on a magnetic stirrer. Then the flask contents were eluted with 20 ml methanol. The collected methanol fractions were evaporated in a rotary evaporator (EYELA, Japan) and the residue was dissolved in DMSO and stored at -20°C for further analysis.
6
Fractionation and Characterization of Natural Compounds
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Diaion HP-20 resin and sephadex LH-20 used for separation were purchased from Sigma-Aldrich (St. Louis, MO, USA) and GE Healthcare (Uppsala, Sweden), respectively. All organic solvents used for extraction and isolation were obtained from Samchun (Pyeongtaek-si, Gyeonggi-do, Korea). Ultrapure water used for extraction, isolation and all solutions was obtained using a Milli-Q laboratory water pufification system (Millipore, Bedford, MA, USA) with a resistivity over 18.2 MΩ·cm. Fourier-transform-NMR spectrometer used for nuclear magnetic resonance were obtained from Bruker Korea (Seongnam, Korea). Signal processing and interpretation were performed using the Bruker DPX 400 MHz (9.4T) package. JMS-700 Mstation used for EI-MS were obtained from JEOL (Tokyo, Japan). Voyager-DE-STR-MALDI-TOF Mass Spectrometer used for MALDI-TOF MS was purchased from Applied Biosysterms (Foster City, CA, USA).
7
Purification and Characterization of Thailanstatins
8
Solid-Phase Peptide Synthesis Protocol
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2-chlorotrityl chloride resin, BOP (benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate), and HOBt.H2O (hydroxybenzotriazole monohydrate) were purchased from Novabiochem (Merck KGaA, Darmstadt, Germany). Boc-Gly-Val was purchased from Bachem (Bachem Americas, Inc., Torrance, USA). D-Glucosamine HCl, Glycylsarcosine (Gly-Sar), NMM (N-methylmorpholine), mannosamine HCl (MA), amantadine HCl (1-aminoadmantane HCl, ADAM), TEA (triethylamine), TFA (trifluoroacetic acid), DPBS (dulbecco's phosphate buffered saline), Diaion HP-20 resin, and Fmoc-Cl (9-fluorenylmethoxycarbonyl chloride) were purchased from Sigma-Aldrich Canada, Ltd, (Oakville, ON, Canada). HPLC grade acetonitrile (ACN), water, dichloromethane (DCM) and dimethylformamide (DMF) were obtained from Caledon Laboratories Ltd, (ON, Canada). All other chemicals and solvents were commercial products of analytical or HPLC grades.
9
Purification of FK228 from Fermentation Broth
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Because FK228 was previously found to present mostly in the aqueous phase, fermentation broth was first centrifuged at 3800 rpm for 20 min to remove cell debris. The resulting supernatant was passed three times through a large glass column (i.d. 10 × 100 cm) packed 1/3 full with a mixture of Amberlite XAD16 resin (20–60 mesh, Sigma-Aldrich, USA) and Diaion HP-20 resin (Sigma-Aldrich) in a 1:1 ratio. Resin mixture was further washed with water, drained and eluted repeatedly with three volumes of ethyl acetate. Elution fractions were pooled and concentrated with a rotary evaporator, and the resulting oily residue was subjected to three steps of chromatographic separation to obtain FK228 with greater than 98% purity (Fig. S2 ).
10
Antimicrobial Potential of Soil and Water Microbes
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We isolated the microbes from two different niches: soil and water samples from Leh and Ladakh, India. The samples, after serial dilution in normal saline, were spread plated on three different media, i.e., R2A agar (HiMedia), tryptic soy agar (HiMedia) and Actinomycete isolation agar (HiMedia) and incubated for 1 week at 30 and 37°C. All purified isolates were preserved in 20% glycerol stock at -80°C. Isolates were grown in tryptic soy broth for 24–96 h and crude extracts were prepared using Diaion HP-20 resin (Sigma) as explained in Section “Purification of Antimicrobial Compound(s) From PV3-16 Strain.” Antimicrobial activity of cell-free supernatants and extracts was assessed using agar well diffusion assay (Valgas et al., 2007 (link)) with the test strain seeded in the molten agar. Test strains used were: E. coli ATCC 25922, Klebsiella pneumoniae ATCC 29665, S. aureus ATCC 25923 and C. albicans ATCC 10231. Positive isolates were identified based on 16S rRNA gene sequencing. The isolate PV3-16, previously unreported for antimicrobial production, was selected for further study.
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