The cDNA library was size-fractionated on a 2% TAE low melt agarose gel (Lonza catalog # 50080), with a 100 bp ladder (Roche catalog # 14703220) run in adjacent lanes. Prior to loading on the gel, the ligated cDNA library was taken over a G50 Sephadex column to remove excess salts that interfere with loading the sample in the wells. After post-staining the gel in ethidium bromide, a narrow slice (∼2mm) of the cDNA lane centered at the 300 bp marker was cut. The slice was extracted using the QiaEx II kit (Qiagen catalog # 20021), and the extract was filtered over a Microcon YM-100 microconcentrator (Millipore catalog # 42409) to remove DNA fragments shorter than 100 bps. Filtration was performed by pipeting the extract into the upper chamber of a microconcentrator, and adding ultra pure water (Gibco catalog # 10977) to a volume of 500 uLs. The filter was spun at 500 X g until only 50 uLs remained in the upper chamber (about 20 minutes per spin) and then the upper chamber volume was replenished to 500 uLs. This procedure was repeated 6 times. The filtered sample was then recovered from the filter chamber according to the manufacturer's protocol. Fragment length distributions obtained after size selection were estimated from the spike-in sequences and are show in Supplementary Fig. 1 .
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Sephadex G 100
Sephadex G 100
Sephadex G 100 is a size-exclusion chromatography medium used for the separation and purification of biomolecules, such as proteins, nucleic acids, and other macromolecules.
It is composed of cross-linked dextran beads with a fractionation range suitable for the analysis of molecules with a molecular weight between 4,000 and 150,000 Daltons.
Sephadex G 100 is commonly employed in various biochemical and biopharmaceutical applications, including protein purification, enzyme studies, and the characterization of protein-protein interactions.
Its versatility and reliable performance make it a valuable tool for researchers in the fields of molecular biology, biotechnology, and biochemistry.
It is composed of cross-linked dextran beads with a fractionation range suitable for the analysis of molecules with a molecular weight between 4,000 and 150,000 Daltons.
Sephadex G 100 is commonly employed in various biochemical and biopharmaceutical applications, including protein purification, enzyme studies, and the characterization of protein-protein interactions.
Its versatility and reliable performance make it a valuable tool for researchers in the fields of molecular biology, biotechnology, and biochemistry.
Most cited protocols related to «Sephadex G 100»
cDNA Library
DNA, Complementary
Ethidium Bromide
Filtration
Salts
sephadex
Sepharose
Previously crystallized GPCRs show little density for the poorly ordered amino and carboxy terminal domains. Although these domains are not critical for maintaining high ligand affinity, these flexible regions may inhibit crystallogenesis7 (link). We therefore removed these regions in the receptor construct used for crystallography. Specifically, a TEV protease recognition site was introduced after reside G51 in the amino-terminus and the carboxy terminus was truncated after Q360. The short third intracellular loop of μOR, consisting of residues 264–269 was replaced with T4 lysozyme residues 1–161 in a manner described previously7 (link). In order to facilitate receptor purification, a FLAG M1 tag was added to the amino-terminus and an octa-histidine tag was appended to the carboxy terminus. Finally, a proline residue was introduced N-terminal to the octahistidine tag to allow efficient removal of C-terminal histidines by carboxypeptidase A. For these studies, we utilized the Mus musculus μOR sequence because it expressed at higher levels. The mouse and human μOR share 94% sequence identity and there are only four residues in the resolved part of the structure that differ between mouse and human μOR. These include residues 66, 137, 187, and 306, which are all in the extracellular or intracellular loops of μOR and do not make contacts in the ligand-binding pocket. The final crystallization construct (μOR-T4L) is shown in a representative snake diagram in Supplementary Fig. 1a .
We compared the pharmacological properties of μOR-T4L to those of the wild-type receptor (Supplementary Fig. 1b ; see below for methods details). Both constructs showed identical affinity for the radiolabeled antagonist [3H]-diprenorphine ([3H]DPN).
The μOR-T4L construct was expressed in Sf9 cells using the baculovirus system. Culture media was supplemented with 10 μM naloxone to stabilize the receptor during expression. Cells were infected at a density of 4×106 cells per mL and culture flasks were shaken at 27 °C for 48 hr. After harvesting, cells were lysed by osmotic shock in a buffer comprised of 10mM Tris-HCl pH 7.5, 1mM EDTA, 100 µM TCEP, 1 µM naloxone, and 2 mg/ml iodoacetamide to block reactive cysteines. Extraction of μOR-T4L from Sf9 membranes was done with a Dounce homogenizer in a solubilization buffer comprised of 0.5% dodecyl maltoside (DDM), 0.3% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.03% cholesterol hemisuccinate (CHS), 20 mM HEPES pH 7.5, 0.5 M NaCl, 30% v/v glycerol, 2 mg/ml iodoacetamide, 100 μM TCEP, and 1 µM naloxone. After centrifugation, nickel-NTA agarose was added to the supernatant, stirred for two hours, and then washed in batch with 100 × g spins for 5 min each with a washing buffer of 0.1% DDM, 0.03% CHAPS, 0.01% CHS, 20 mM HEPES pH 7.5 and 0.5 M NaCl. The resin was poured into a glass column and bound receptor was eluted in washing buffer supplemented with 300 mM imidazole.
We utilized anti-FLAG M1 affinity resin to further purify μOR-T4L and to exchange the ligand to the covalent antagonist β-funaltrexamine (β-FNA). Nickel-resin eluate was loaded onto anti-FLAG M1 resin and washed extensively in the presence of 10 µM β-FNA. The detergent DDM was then gradually exchanged over 1 hr into a buffer with 0.01% lauryl maltose neopentyl glycol (MNG) and the NaCl concentration was lowered to 100 mM. Receptor was eluted from the anti-FLAG M1 affinity resin with 0.2 mg/mL FLAG peptide and 5 mM EDTA in the presence of 1 µM β-FNA. To remove the amino terminus of μOR-T4L, TEV protease was added at 1:3 w/w (TEV:μOR-T4L) and incubated at room temperature for 1 hr. Receptor was then treated with carboxypeptidase A (1:100 w/w) and incubated overnight at 4°C to remove the octa-histidine tag. The final purification step separated TEV and carboxypeptidase A from receptor by size exclusion chromatography (SEC) on a Sephadex S200 column (GE Healthcare) in a buffer of 0.01% MNG, 0.001% CHS, 100 mM NaCl, 20 mM HEPES pH 7.5, and 1 µM β-FNA. After size exclusion, β-FNA was added to a final concentration of 10 µM. The resulting receptor preparation was pure and monodisperse (Supplementary Fig. 12 ).
We compared the pharmacological properties of μOR-T4L to those of the wild-type receptor (
The μOR-T4L construct was expressed in Sf9 cells using the baculovirus system. Culture media was supplemented with 10 μM naloxone to stabilize the receptor during expression. Cells were infected at a density of 4×106 cells per mL and culture flasks were shaken at 27 °C for 48 hr. After harvesting, cells were lysed by osmotic shock in a buffer comprised of 10mM Tris-HCl pH 7.5, 1mM EDTA, 100 µM TCEP, 1 µM naloxone, and 2 mg/ml iodoacetamide to block reactive cysteines. Extraction of μOR-T4L from Sf9 membranes was done with a Dounce homogenizer in a solubilization buffer comprised of 0.5% dodecyl maltoside (DDM), 0.3% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.03% cholesterol hemisuccinate (CHS), 20 mM HEPES pH 7.5, 0.5 M NaCl, 30% v/v glycerol, 2 mg/ml iodoacetamide, 100 μM TCEP, and 1 µM naloxone. After centrifugation, nickel-NTA agarose was added to the supernatant, stirred for two hours, and then washed in batch with 100 × g spins for 5 min each with a washing buffer of 0.1% DDM, 0.03% CHAPS, 0.01% CHS, 20 mM HEPES pH 7.5 and 0.5 M NaCl. The resin was poured into a glass column and bound receptor was eluted in washing buffer supplemented with 300 mM imidazole.
We utilized anti-FLAG M1 affinity resin to further purify μOR-T4L and to exchange the ligand to the covalent antagonist β-funaltrexamine (β-FNA). Nickel-resin eluate was loaded onto anti-FLAG M1 resin and washed extensively in the presence of 10 µM β-FNA. The detergent DDM was then gradually exchanged over 1 hr into a buffer with 0.01% lauryl maltose neopentyl glycol (MNG) and the NaCl concentration was lowered to 100 mM. Receptor was eluted from the anti-FLAG M1 affinity resin with 0.2 mg/mL FLAG peptide and 5 mM EDTA in the presence of 1 µM β-FNA. To remove the amino terminus of μOR-T4L, TEV protease was added at 1:3 w/w (TEV:μOR-T4L) and incubated at room temperature for 1 hr. Receptor was then treated with carboxypeptidase A (1:100 w/w) and incubated overnight at 4°C to remove the octa-histidine tag. The final purification step separated TEV and carboxypeptidase A from receptor by size exclusion chromatography (SEC) on a Sephadex S200 column (GE Healthcare) in a buffer of 0.01% MNG, 0.001% CHS, 100 mM NaCl, 20 mM HEPES pH 7.5, and 1 µM β-FNA. After size exclusion, β-FNA was added to a final concentration of 10 µM. The resulting receptor preparation was pure and monodisperse (
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Fluxes of H+ or Cl− were followed by continuous recording, with ion-specific electrodes, in suspensions of liposomes reconstituted with CLC-ec1. Voltage from the electrodes was fed to an Orion 701A high-sensitivity pH meter (Ebay.com) and digitized at 5–10 Hz by a DI-70 datalogger (DATAQ Instruments). Inward proton pumping driven by an outward Cl− gradient was assayed as described previously (Accardi and Miller, 2004 (link)), using a glass pH electrode to follow H+ uptake in a lightly buffered suspension. After thawing, liposomes (2.5 μg/mg protein density) loaded with 300 mM KCl, 25 mM CPi, pH 4.8, were extruded 21 times through a 400-nm membrane filter (Nguitragool and Miller, 2006 (link)) and were then centrifuged through Sephadex G-50 (100-μl sample per 1.5 ml column) equilibrated with proton-pumping buffer (PPB), 290 mM K-isethionate, 10 mM KCl, 2 mM citrate, pH 5.2, and diluted 10-fold into PPB in a 2-ml stirred cell fitted with a pH electrode. Proton uptake was initiated by addition of 1 μM valinomycin (Vln) and collapsed by FCCP (2 μM). Proton efflux experiments were set up analogously, using liposomes loaded with 300 mM KCl, 25 mM citrate/25 mM MES, pH 4.5, and suspended in 300 mM KCl, 1 mM citrate/MES pH 6.5.
Net Cl− efflux was similarly followed with Ag/AgCl electrodes in a stirred cell temperature-controlled to 25°C. Electrodes were constructed from silver wire cleaned overnight in concentrated HNO3 and coated with AgCl by immersion in Clorox bleach or 0.1 M FeCl3 solution. Liposomes reconstituted with 0.03–4 μg/mg CLC-ec1, and loaded with 300 mM KCl, 25 mM citrate-NaOH, pH 4.5, were extruded and centrifuged through Sephadex G-50 equilibrated in Cl− dump-buffer (CDB), 300 mM K-isethionate, 1 mM KCl, 25 mM citrate, pH 4.5. The sample containing 1.2 mg lipid was added, and KCl efflux was evoked by Vln/FCCP. After 1–3 min, 50 mM octylglucoside detergent was added to release all trapped Cl−. The electrode voltage signal, V(t), zeroed before initiating the efflux, was converted to the increase in Cl− concentration, Δc(t), above the initial concentration c(0) by: and α, an electrode-imperfection factor (of unknown origin) determined by calibrating with 75 μM Cl− at the beginning of each experiment, falls in the range 0.93 ± 0.03. This time course was fit to a two-component relaxation, one for the fraction (1 − fo) of liposomes containing transporters, the other for the fraction (fo) devoid of protein: where ΔcT, the total concentration of Cl− released in the experiment (determined directly by detergent addition), typically reflects an increase of 0.15–0.2 mM over the 1 mM Cl− present before the efflux. Here, kt and kL are the rate constants for Cl− flux through the transporter and for the background leak through the liposome membrane, respectively. This background leak was measured in separate experiments on protein-free liposomes to be 5.7 ± 0.5 × 10−4 s−1, typically 50-fold lower than the transporter-mediated rate constant. For reasons explained in the text, we report the inverse of kt as the useful transporter-mediated kinetic parameter, denoted the “average time constant,” 〈τ〉. Experiments were temperature controlled at 25°C.
Net Cl− efflux was similarly followed with Ag/AgCl electrodes in a stirred cell temperature-controlled to 25°C. Electrodes were constructed from silver wire cleaned overnight in concentrated HNO3 and coated with AgCl by immersion in Clorox bleach or 0.1 M FeCl3 solution. Liposomes reconstituted with 0.03–4 μg/mg CLC-ec1, and loaded with 300 mM KCl, 25 mM citrate-NaOH, pH 4.5, were extruded and centrifuged through Sephadex G-50 equilibrated in Cl− dump-buffer (CDB), 300 mM K-isethionate, 1 mM KCl, 25 mM citrate, pH 4.5. The sample containing 1.2 mg lipid was added, and KCl efflux was evoked by Vln/FCCP. After 1–3 min, 50 mM octylglucoside detergent was added to release all trapped Cl−. The electrode voltage signal, V(t), zeroed before initiating the efflux, was converted to the increase in Cl− concentration, Δc(t), above the initial concentration c(0) by: and α, an electrode-imperfection factor (of unknown origin) determined by calibrating with 75 μM Cl− at the beginning of each experiment, falls in the range 0.93 ± 0.03. This time course was fit to a two-component relaxation, one for the fraction (1 − fo) of liposomes containing transporters, the other for the fraction (fo) devoid of protein: where ΔcT, the total concentration of Cl− released in the experiment (determined directly by detergent addition), typically reflects an increase of 0.15–0.2 mM over the 1 mM Cl− present before the efflux. Here, kt and kL are the rate constants for Cl− flux through the transporter and for the background leak through the liposome membrane, respectively. This background leak was measured in separate experiments on protein-free liposomes to be 5.7 ± 0.5 × 10−4 s−1, typically 50-fold lower than the transporter-mediated rate constant. For reasons explained in the text, we report the inverse of kt as the useful transporter-mediated kinetic parameter, denoted the “average time constant,” 〈τ〉. Experiments were temperature controlled at 25°C.
Genomic DNA was extracted using the Ultraclean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. Parts of the following loci were amplified and sequenced for the species listed in Table 1 : 1. RPB1, RNA polymerase II largest subunit (regions E and F; according Matheny et al. 2002 (link)), 2. RPB2, RNA polymerase II second largest subunit (regions 5–7), 3. Cct8, subunit of the cytosolic chaperonin Cct ring complex, related to Tcp1p and required for the assembly of actin and tubulins in vivo (Stoldt et al. 1996 (link), Kim et al. 1994 (link)), 4. Tsr1, protein required for processing of 20S pre-rRNA in the cytoplasm (Gelperin et al. 2001 (link), Léger-Silvestre et al. 2004 (link)). Partial RPB2 data was obtained for the majority of species listed in Table S1. Exceptions are strains used in the study of Houbraken et al. (2011c (link)); in that case, published partial β-tubulin sequences were used.
The RPB1 fragment was amplified using the primer pair RPB1-F1843 and R3096, and RPB1-R2623 was occasionally used as an internal primer for sequencing. A part of the RPB2 locus was amplified using the primer pair RPB2-5F and RPB2-7CR (Liu et al. 1999 (link)) or the primer pair RPB2-5F_Eur and RPB2-7CR_Eur. The internal sequencing primers RPB2-F311 and RPB2-R310 were occasionally used when poor results were obtained with the regular forward and reverse primers. Amplification of a part of the Cct8 gene was performed using the primer pair Cct8-F660 and Cct8-R1595. No amplicons could be obtained in the case of 5–10 % of the analysed strains. In those cases, amplicons were generated using the primer pair Cct8-R1595 and Cct8-F94. A part of the Tsr1 gene was amplified using the forward primers Tsr1-F1526Pc or Tsr1-F1526 in combination with Tsr1-R2434. Annealing temperatures and primers used for amplification and sequencing are shown inTable 2 .
The PCR reactions were performed in 25 μL reaction mixtures containing 1 μL genomic DNA 2.5 μL PCR buffer, 0.75 μL MgCl2 (50 mM), 16.55 μL demineralised sterile water, 1.85 μL dNTP (1 mM), 0.50 μL of each primer (100 mM) and 0.1 μL Taq polymerase (5 U/μL, BioTaq, Bioline). The PCR program typically was: 5 cycles of 30 s denaturation at 94 °C, followed by primer annealing for 30 s at 51 °C, and extension for 1 min at 72 °C; followed by 5 cycles with an annealing temperature at 49 °C and 30 cycles at 47 °C, finalised with an extension for final 10 min at 72 °C. Excess primers and dNTP's were removed from the PCR product using the QIAQuick PCR purification kit (Qiagen). Purified PCR fragments were resuspended in 30–50 μL of water. PCR products were sequenced directly in both directions with the same primers and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience, Roosendaal, The Netherlands). The cycle sequencing reaction mixture had a total reaction volume of 10 μL, and contained 1 μL of template DNA, 0.85 μL BigDye reagent, 3 μL buffer, 4.75 μL demineralised water and 0.4 μL primer (10 mM).
Sequencing products were purified according to the manufacturers' recommendations with Sephadex G-50 superfine columns (Amersham Bioscience, Roosendaal, The Netherlands) in a multiscreen HV plate (Millipore, Amsterdam, The Netherlands) and with MicroAmp Optical 96-well reaction plate (AB Applied Biosystems, Nieuwerkerk a/d Yssel, The Netherlands). Contigs were assembled using the forward and reverse sequences with the programme SeqMan from the LaserGene package (DNAStar Inc., Madison, WI).
The RPB1 fragment was amplified using the primer pair RPB1-F1843 and R3096, and RPB1-R2623 was occasionally used as an internal primer for sequencing. A part of the RPB2 locus was amplified using the primer pair RPB2-5F and RPB2-7CR (Liu et al. 1999 (link)) or the primer pair RPB2-5F_Eur and RPB2-7CR_Eur. The internal sequencing primers RPB2-F311 and RPB2-R310 were occasionally used when poor results were obtained with the regular forward and reverse primers. Amplification of a part of the Cct8 gene was performed using the primer pair Cct8-F660 and Cct8-R1595. No amplicons could be obtained in the case of 5–10 % of the analysed strains. In those cases, amplicons were generated using the primer pair Cct8-R1595 and Cct8-F94. A part of the Tsr1 gene was amplified using the forward primers Tsr1-F1526Pc or Tsr1-F1526 in combination with Tsr1-R2434. Annealing temperatures and primers used for amplification and sequencing are shown in
The PCR reactions were performed in 25 μL reaction mixtures containing 1 μL genomic DNA 2.5 μL PCR buffer, 0.75 μL MgCl2 (50 mM), 16.55 μL demineralised sterile water, 1.85 μL dNTP (1 mM), 0.50 μL of each primer (100 mM) and 0.1 μL Taq polymerase (5 U/μL, BioTaq, Bioline). The PCR program typically was: 5 cycles of 30 s denaturation at 94 °C, followed by primer annealing for 30 s at 51 °C, and extension for 1 min at 72 °C; followed by 5 cycles with an annealing temperature at 49 °C and 30 cycles at 47 °C, finalised with an extension for final 10 min at 72 °C. Excess primers and dNTP's were removed from the PCR product using the QIAQuick PCR purification kit (Qiagen). Purified PCR fragments were resuspended in 30–50 μL of water. PCR products were sequenced directly in both directions with the same primers and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Bioscience, Roosendaal, The Netherlands). The cycle sequencing reaction mixture had a total reaction volume of 10 μL, and contained 1 μL of template DNA, 0.85 μL BigDye reagent, 3 μL buffer, 4.75 μL demineralised water and 0.4 μL primer (10 mM).
Sequencing products were purified according to the manufacturers' recommendations with Sephadex G-50 superfine columns (Amersham Bioscience, Roosendaal, The Netherlands) in a multiscreen HV plate (Millipore, Amsterdam, The Netherlands) and with MicroAmp Optical 96-well reaction plate (AB Applied Biosystems, Nieuwerkerk a/d Yssel, The Netherlands). Contigs were assembled using the forward and reverse sequences with the programme SeqMan from the LaserGene package (DNAStar Inc., Madison, WI).
Most recents protocols related to «Sephadex G 100»
After mixing with silica 60 RP-C18, the MSA fraction (23.9 g) was placed on an RP-C18 column (50 g) and eluted with H2O/MeOH (100:0 → 0:100, v/v) to obtain 12 fractions (Fr.1–Fr.12). Fr.9 (1.617 g) was separated with a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain nine fractions (Fr.9.1-9). Fr.9.3 (1.254 g) was separated using a silica gel column (200–300 mesh) and eluted with petroleum ether/acetone (100:1 → 6:4, v/v) to obtain five fractions (Fr.9.3.1-5). Fr.9.3.2 (135 mg) was loaded on a silica gel column eluting with chloroform/acetone (100:1 → 6:4, v/v) and then purified by Sephadex LH-20 (acetone) to obtain compound 8 (3 mg). Fr.9.3.4 (180 mg) was subjected to Sephadex LH-20 chromatography (chloroform–methanol, 1:1) to obtain four fractions (Fr.9.3.4.1-4). Fr.9.3.4.3 (132 mg) underwent separation on a silica gel column eluted with chloroform/acetone (100:1 → 8:2, v/v) and was subsequently purified by a Sephadex LH-20 (acetone) to obtain compound 9 (2 mg). Fr.9.3.5 (113 mg) was loaded on a Sephadex LH-20 (acetone) and purified on a silica gel column via elution with chloroform/methanol (100:1 → 8:2, v/v) to obtain compound 10 (8 mg). Fr.5 (259 mg) was subjected to Sephadex LH-20 (chloroform–methanol, 1:1) chromatography to obtain five fractions (Fr.5.1-5). Fr.5.4 (54 mg) was further separated using a silica gel column via elution with chloroform/acetone (100:1 → 6:4, v/v) and purified by Sephadex LH-20 chromatography (acetone) to obtain compound 2 (7 mg). Fr.5.5 (80 mg) was subjected to silica gel column chromatography and eluted with chloroform/acetone (100:0 → 8:2, v/v) and purified by Sephadex LH-20 chromatography (methanol) to obtain compound 7 (5 mg). Fr.4 (1.6 g) was loaded on a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain seven fractions (Fr.4.1-7). Fr.4.5 (637 mg) was further separated using Sephadex LH-20 (methanol) to obtain four fractions (Fr.4.5.1-4). Fr.4.5.2 (439 mg) was subjected to semi-preparative HPLC with gradient elution of MeOH–H2O (30:70 → 35:65 and 40:60 → 100:0) for 50 min to obtain five fractions (Fr.4.5.2.1-5). Fr.4.5.2.3 (120 mg) was separated using a Sephadex LH-20 column (methanol) to obtain three fractions (Fr.4.5.2.3.1-3). Fr.4.5.2.3.3 (108 mg) was further separated using a silica gel column via elution with chloroform/acetone (100:1 → 0:100, v/v) to obtain eight fractions (Fr.4.5.2.3.3.1-8). Fr.4.5.2.3.3.6 (51 mg) was separated using a Sephadex LH-20 column (methanol) to obtain compound 6 (28 mg). Fr.4.5.2.5 (64 mg) was separated using a Sephadex LH-20 column (methanol) to obtain two fractions (Fr.4.5.2.5.1-2). Fr.4.5.2.5.2 (59 mg) was further separated using a column of silica gel via elution with chloroform/acetone (100:1 → 0:100, v/v) and purified by Sephadex LH-20 (methanol) to obtain compound 3a/b (5 mg). Fr.3 (2.135 g) was separated using a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain five fractions (Fr.3.1-5). Fr.3-5 (1.232 g) was separated using a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain four fractions (Fr.3.5.1-4). Fr.3.5.4 (1.081 g) was subjected to semi-preparative RP-C18 HPLC with a gradient elution of MeOH:H2O (10:90 → 45:55 and 60:40 → 100:0) for 40 min to obtain three fractions (Fr.3.5.4.1-3). Fr.3.5.4.3 (34 mg) was further separated using a silica gel column via elution with chloroform/acetone (50:1 → 0:100, v/v) to obtain four fractions (Fr.3.5.4.3.1-4). Fr.3.5.4.3.2 (8 mg) was separated using a Sephadex LH-20 column (methanol) to obtain compound 4 (5 mg).
The WB fraction (30.0 g) was placed on an RP-C18 column (60 g) and eluted with H2O/MeOH mixtures (100:0 → 0:100, v/v) to obtain 15 fractions (Fr.1-15). Fr.15 (878 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (300:1 → 6:4, v/v) to obtain five fractions (Fr.15.1-5). Fr.15.2 (153 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (100:1 → 8:2, v/v) and then purified with a silica gel column via elution with petroleum ether/ethyl acetate (200:1 → 8:2, v/v) to obtain compound11 (1 mg). Fr.15.4 (123 mg) was separated using a silica gel column via elution with chloroform/acetone (10:1 → 6:4, v/v) and then purified by Sephadex LH-20 (acetone) to obtain compound 8 (2 mg). Fr.6 (385 mg) was loaded on a Sephadex LH-20 column (methanol) to obtain six fractions (Fr.6.1-6). Fr.6.3 (108 mg) was subjected to a silica gel column (100:1 → 7:3, v/v) and purified by Sephadex LH-20 (methanol) to obtain compound 1 (6 mg). Fr.4 (1.15 g) was subjected to Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain five fractions (Fr.4.1-5). Fr.4.1 (715 mg) was loaded on a silica gel column via elution with chloroform/acetone (100:1 → 8:2, v/v) to obtain four fractions (Fr.4.1-4). Fr.4.1.3 (83 mg) was subjected to a Sephadex LH-20 column (methanol) and purified by a silica gel column via elution with chloroform/acetone (100:1 → 8:2, v/v) to obtain compound 5 (15 mg).
The WB fraction (30.0 g) was placed on an RP-C18 column (60 g) and eluted with H2O/MeOH mixtures (100:0 → 0:100, v/v) to obtain 15 fractions (Fr.1-15). Fr.15 (878 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (300:1 → 6:4, v/v) to obtain five fractions (Fr.15.1-5). Fr.15.2 (153 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (100:1 → 8:2, v/v) and then purified with a silica gel column via elution with petroleum ether/ethyl acetate (200:1 → 8:2, v/v) to obtain compound
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Example 1
Initial evaluation of the hemostatic properties of a composition comprising an anion exchanger and a calcium salt was carried out in an in-vivo heparinized porcine spleen circular punch model as described above, using DEAE covalently bound to Sephadex (DEAE-SEPHADEX™) as the anion exchanger. In this experiment, the punch size was 4 mm diameter, 2 mm depth. A compression time of 30 or 60 seconds was used following application. DEAE SEPHADEX™ A-50 was tested at two concentrations. In this experiment, the Post-Application Bleeding Intensity was evaluated qualitatively.
The following compositions (see elaboration in Table 1 above) were evaluated for their hemostatic efficacy:
-
- 1. DEAE SEPHADEX™ A-50, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg DEAE SEPHADEX™ A-50) (30 seconds compression time);
- 2. DEAE SEPHADEX™ A-50, prepared as 6.6% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 33 mg DEAE SEPHADEX™ A-50) (60 seconds compression time);
- 3. SEPHADEX™ G-75 Superfine, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg per SEPHADEX™ G-75 Superfine) (60 seconds compression time);
- 4. SEPHADEX™ G-50 Medium, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg SEPHADEX™ G-50 Medium) (60 seconds compression time);
- 5. Commercial gelatin hemostat prepared as a slurry (0.5 ml contains 55 mg gelatin) (60 seconds compression time).
All four SEPHADEX™ samples comprise the same base polymer, cross-linked dextran. Compositions 1-4 were provided as powders, from which slurries were prepared as described in the Table 1 above. A commercial gelatin flowable hemostat was used as control.
It was found that SEPHADEX™ G-50 Medium, SEPHADEX™ G-75 Superfine and commercial gelatin hemostat, failed to stop the bleeding, i.e. no reduction in bleeding intensity was observed (results not shown).
Surprisingly, DEAE SEPHADEX™ A-50 reduced the bleeding at all tested compression times. Following the application of DEAE SEPHADEX™ A-50, the spleen was manually manipulated by folding the organ from both sides. No re-bleeding occurred at either of the tested concentrations and following the two different compression times (results not shown). Since hemostasis only occurred in the matrix supplemented with DEAE groups it was concluded that the hemostatic effect was due the presence of the DEAE groups.
Example 2
In the following Example, the effect on hemostasis of each of the components of a composition comprising an anion exchanger and a calcium salt was evaluated, separately and in combination, using an in-vivo heparinized porcine liver circular punch model, as described above. This experiment identifies which of the components of the composition are required for achieving hemostasis.
The preparation of each composition is described in Table 1 above. Compression time is listed in Table 2 below. In this experiment, the Initial Bleeding Intensity and Post-Application Bleeding Intensity were evaluated according to the 0-5 scale.
The following compositions were evaluated:
-
- 1. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);
- 2. Commercial gelatin hemostat, prepared as a slurry (0.5 ml contains 55 mg gelatin);
- 3. Commercial gelatin hemostat with thrombin, prepared as a slurry (0.5 ml contains 55 mg gelatin);
- 4. SEPHADEX™ G-50 Medium, prepared as 14% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 70 mg SEPHADEX™ G-50 Medium).
- 5. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM NaCl solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);
- 6. SP SEPHADEX™ C-50, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg SP SEPHADEX™ C-50);
- 7. QAE SEPHADEX™, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg QAE SEPHADEX™);
- 8. DEAE SEPHACEL™, prepared as a slurry (100 mg); and
- 9. TOYOPEARL DEAE-650M™, prepared in powder form (100 mg).
The compression time following the application of each tested composition, and the bleeding intensity results are shown in Table 2. Bleeding Intensity Reduction was calculated by subtracting the Post-Application Bleeding Intensity from the Initial Bleeding Intensity.
In general, it can be seen, that a composition comprising an anion exchanger, such as DEAE bound to a matrix, together with a calcium salt, lead to complete hemostasis (see Table 2 for DEAE SEPHADEX™ A-50, DEAE SEPHACEL™, and TOYOPEARL DEAE-650M™, all containing a calcium salt). These compositions substantially lead to complete hemostasis regardless of the specific matrix used. For example, matrices such as SEPHADEX™, SEPHACEL™ and TOYOPEARL™ (dextran, cellulose and hydroxylated methacrylic polymer, respectively) had a similar effect in reducing the bleeding intensity.
More particularly, DEAE SEPHADEX™ A-50 8% w/v in CaCl2 was able cease bleeding after 60, and 30 seconds of compression. The results also showed DEAE SEPHADEX™ A-50 (8% w/v) application could be used, without compression, to reduce bleeding intensity (Table 2).
The hemostatic capabilities of a composition comprising DEAE SEPHADEX™ A-50 and a calcium salt exhibited similar efficacy to that of commercial gelatin hemostat with thrombin, when using the same compression time (30 seconds), and even with only 10 seconds of compression. However, the hemostatic capability of a hemostat based on an anion exchanger comprising DEAE bound to a matrix, and a calcium salt, was substantially superior to that of commercial gelatin hemostat in the absence of a biologically active component, such as thrombin.
As shown in Table 3, a composition comprising DEAE SEPHADEX™ prepared with NaCl, and lacking a calcium salt produced no reduction in bleeding intensity, such that it can be concluded that the sample was not effective in stopping the bleeding.
When evaluating the impact of the ion exchange group on the hemostatic capability, it was shown that SP SEPHADEX™ containing an anionic group, sulfopropyl (SP) with a calcium salt, did not reduce the bleeding intensity. In other words, a material with a negative (SP) group, instead of a positive (DEAE) group was not effective as a hemostat.
It was further shown that a quaternary aminoethyl, QAE SEPHADEX™ with a calcium salt was able to reduce bleeding intensity.
It was also shown, as in Example 1, that the matrix alone, in the absence of a functional group (SEPHADEX™ G-50 with a calcium salt but without DEAE groups) had no hemostatic efficacy.
The results show that use of a composition comprising DEAE groups bound to a matrix in presence of a calcium salt effectively achieved hemostasis.
The results also showed that compression time of 30 and 60 seconds following DEAE SEPHADEX™ A-50 (8% w/v) application resulted in complete hemostasis and therefore the TTH was defined as 30 seconds.
It was thus shown that an anion exchanger such as DEAE bound to a matrix together with a calcium salt provided complete hemostasis. This result was obtained regardless of the matrix used. The results are comparable to those obtained when using a commercial hemostat such as gelatin with thrombin.
It was found that QAE SEPHADEX™ together with a calcium salt reduced bleeding.
A composition devoid of a calcium salt and/or a matrix had no effect on the bleeding intensity.
These results suggest that a composition comprising an anion exchanger and a calcium salt is effective as a hemostat.
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Dried whole plants of C. fuscescens Lindl. var.brunnea (2.3 kg) were macerated with ethanol (EtOH) (5 × 10 L). The
ethanolic extract (530 g) was mixed with water and subjected to partitioning
with ethyl acetate (EtOAc). This process yielded an ethyl acetate
extract (99.2 g) and an aqueous extract (428.2 g).
The ethyl
acetate extract was then separated by vacuum liquid chromatography
(silica gel, EtOAc-hexane, gradient) to yield nine fractions (F1–F9).
F4 (7.8 g) was subjected to further fractionation on a silica gel
column (EtOAc-hexane, gradient) to yield compound5 (50
mg). Fraction F6 (5 g) was separated on a silica gel column (EtOAc-hexane,
gradient), resulting in three fractions (F6A–F6C), and fraction
F6B (1 g) was then subjected to CC using an EtOAc-hexane gradient
to yield three fractions (F6BA–F6BC). F6BA (150 mg) was separated
again on CC (silica gel, EtOAc-hexane, gradient) to give compounds6 (7.8 mg) and 7 (2.8 mg). F6BB (250 mg) was
separated by CC (silica gel, EtOAc-hexane, and gradient) to produce
compounds8 (29.3 mg) and 9 (14 mg). F6BC
(500 mg) was subjected to CC (silica gel, EtOAc-hexane, and gradient)
to produce compounds10 (37.1 mg) and 11 (120.9 mg). Fraction F7 (35 g) was fractionated on a silica gel
column (EtOAc-hexane, gradient), and four fractions (F7A–F7D)
were obtained. Fraction F7D (155 mg) was purified again by Sephadex
LH-20 (acetone) to give three fractions (F7D1–F7D3). Fraction
F7D3 (34 mg) was then separated on silica gel (EtOH-toluene, 0.5:9.5)
to furnish compound12 (14 mg). Fraction F8 (5 g) was
then separated by CC on silica gel with a gradient of EtOH–dichloromethane
to yield four fractions (F8A–F8D). F8B (800 mg) was separated
again by Sephadex LH-20 (acetone) to yield three fractions (F8B1–F8B3).
F8B2 (300 mg) was further separated on a silica gel column (EtOH-dichloromethane,1:9),
and three fractions (F8B2A–F8B2C) were obtained. F8B2A (50
mg) was separated again by HPLC using the reverse phase (MeOH–H2O, 1:1) to yield compound1 (2.3 mg). F8C (450
mg) was isolated again by Sephadex LH-20 (methanol) to give four fractions
(F8C1–F8C4). F8C2 (100 mg) is separated again with a silica
gel column (EtOH-dichloromethane,1:9) to yield four fractions (F8C2A–F8C2D).
Compound2 (1.9 mg) was obtained from the fraction F8C2C
(30 mg) by Sephadex LH-20 (acetone). F8C3 (100 mg) was separated on
CC (silica gel, EtOH-dichloromethane,1:9) to produce four fractions
(F8C3A–F8C3D). F8C3B (50 mg) was separated by Sephadex LH-20
(MeOH), and compound4 (17.7 mg) was obtained. Separation
of fraction F8D (899 mg) was done by reverse-phase column chromatography
(RP-18), with gradient elution of MeOH (0–100%) in H2O to give four fractions (F8D1–F8D4). F8D2 was separated again
by Sephadex LH-20 (MeOH) to produce three fractions (F8D2A–F8D2C).
F8D2B (8.5 mg) was separated by HPLC using reverse-phase (MeOH–H2O, 1:1) and normal-phase preparative thin-layer chromatography
using mobile phase (EtOH-dichloromethane, 2:8) to furnish compound13 (3.5 mg). F8D4 (20.7 mg) was separated by CC (silica gel,
EtOH-dichloromethane, and gradient) to produce three fractions (F8D4A–F8D4C).
Compound14 (3.5 mg) was obtained by purification of
F8D4B (6 mg) with Sephadex LH-20 (MeOH).
The aqueous extract
(428.2 mg) was separated with Diaion HP-20
(MeOH–H2O, gradient) to give five fractions (AA–AE).
Fraction AB (2 g) is separated again on a silica gel column (EtOH-dichloromethane,
3:7), and five fractions (ABA–ABE) were obtained. Compound15 (68.4 mg) was produced by the purification of the ABE fraction
(200 mg) with Sephadex LH-20 (MeOH). Fraction AC (1 g) was purified
again by Sephadex LH-20 (MeOH) to give four fractions (ACA–ACD).
ACB (200 mg) was separated by CC (silica gel, EtOH-dichloromethane,
3:7) to produce three fractions (ACB1–ACB3). ACB2 (36.8 mg)
was subjected to semipreparative HPLC (silica gel) using the mobile
phase (MeOH-dichloromethane, 1:9), and compound3 (4.3
mg) was furnished.
ethanolic extract (530 g) was mixed with water and subjected to partitioning
with ethyl acetate (EtOAc). This process yielded an ethyl acetate
extract (99.2 g) and an aqueous extract (428.2 g).
The ethyl
acetate extract was then separated by vacuum liquid chromatography
(silica gel, EtOAc-hexane, gradient) to yield nine fractions (F1–F9).
F4 (7.8 g) was subjected to further fractionation on a silica gel
column (EtOAc-hexane, gradient) to yield compound
mg). Fraction F6 (5 g) was separated on a silica gel column (EtOAc-hexane,
gradient), resulting in three fractions (F6A–F6C), and fraction
F6B (1 g) was then subjected to CC using an EtOAc-hexane gradient
to yield three fractions (F6BA–F6BC). F6BA (150 mg) was separated
again on CC (silica gel, EtOAc-hexane, gradient) to give compounds
separated by CC (silica gel, EtOAc-hexane, and gradient) to produce
compounds
(500 mg) was subjected to CC (silica gel, EtOAc-hexane, and gradient)
to produce compounds
column (EtOAc-hexane, gradient), and four fractions (F7A–F7D)
were obtained. Fraction F7D (155 mg) was purified again by Sephadex
LH-20 (acetone) to give three fractions (F7D1–F7D3). Fraction
F7D3 (34 mg) was then separated on silica gel (EtOH-toluene, 0.5:9.5)
to furnish compound
then separated by CC on silica gel with a gradient of EtOH–dichloromethane
to yield four fractions (F8A–F8D). F8B (800 mg) was separated
again by Sephadex LH-20 (acetone) to yield three fractions (F8B1–F8B3).
F8B2 (300 mg) was further separated on a silica gel column (EtOH-dichloromethane,1:9),
and three fractions (F8B2A–F8B2C) were obtained. F8B2A (50
mg) was separated again by HPLC using the reverse phase (MeOH–H2O, 1:1) to yield compound
mg) was isolated again by Sephadex LH-20 (methanol) to give four fractions
(F8C1–F8C4). F8C2 (100 mg) is separated again with a silica
gel column (EtOH-dichloromethane,1:9) to yield four fractions (F8C2A–F8C2D).
Compound
(30 mg) by Sephadex LH-20 (acetone). F8C3 (100 mg) was separated on
CC (silica gel, EtOH-dichloromethane,1:9) to produce four fractions
(F8C3A–F8C3D). F8C3B (50 mg) was separated by Sephadex LH-20
(MeOH), and compound
of fraction F8D (899 mg) was done by reverse-phase column chromatography
(RP-18), with gradient elution of MeOH (0–100%) in H2O to give four fractions (F8D1–F8D4). F8D2 was separated again
by Sephadex LH-20 (MeOH) to produce three fractions (F8D2A–F8D2C).
F8D2B (8.5 mg) was separated by HPLC using reverse-phase (MeOH–H2O, 1:1) and normal-phase preparative thin-layer chromatography
using mobile phase (EtOH-dichloromethane, 2:8) to furnish compound
EtOH-dichloromethane, and gradient) to produce three fractions (F8D4A–F8D4C).
Compound
F8D4B (6 mg) with Sephadex LH-20 (MeOH).
The aqueous extract
(428.2 mg) was separated with Diaion HP-20
(MeOH–H2O, gradient) to give five fractions (AA–AE).
Fraction AB (2 g) is separated again on a silica gel column (EtOH-dichloromethane,
3:7), and five fractions (ABA–ABE) were obtained. Compound
(200 mg) with Sephadex LH-20 (MeOH). Fraction AC (1 g) was purified
again by Sephadex LH-20 (MeOH) to give four fractions (ACA–ACD).
ACB (200 mg) was separated by CC (silica gel, EtOH-dichloromethane,
3:7) to produce three fractions (ACB1–ACB3). ACB2 (36.8 mg)
was subjected to semipreparative HPLC (silica gel) using the mobile
phase (MeOH-dichloromethane, 1:9), and compound
mg) was furnished.
P. lurida araC-PoprL was fermented in KB medium and cultured in a shake flask at 28 ℃ and 180 rpm for 4 d, with a total fermentation of 100 L. The fermentation broth was concentrated under reduced pressure and extracted with ethyl acetate to obtain 108.49 g of extract.
The extract was subjected to silica gel G column chromatography (CC) and eluted with petroleum ether/ ethyl acetate (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 0:1, v/v), ethyl acetate/methanol (60:1, 40:1, 20:1, 10:1, 4:1, 1:1, 0:1, v/v) and pure methanol in turn to obtain 21 components, E1-E21. Fraction E2 was subjected to Sephadex LH-20 CC with acetone to obtain three fractions, E2-1-to E2-3. Fraction E2-3 was subjected to silica gel G CC eluted with petroleum ether/acetone/ formic acid (1000:10:1, 800:10:0.8, v/v) to obtain compound 2 (4.7 mg). Fraction E4 was subjected to Sephadex LH-20 CC with methanol gel to obtain fractions E4-1-to E4-7. Fraction E4-5 was isolated through silica gel G CC eluted with petroleum ether/acetone (100:1, 80:1, v/v) to obtain compound 3 (4.2 mg). Fraction E6 was separated by Sephadex LH-20 CC with methanol to obtain fractions E6-1-to E6-3, and fraction E6-2 was subjected to silica gel G CC eluted with petroleum ether/ethyl acetate (100:1, 90:1, 80:1, 70:1, 60:1, v/v) to afford compound 4 (3.3 mg). Fraction E8 was separated by preparative liquid chromatography [Hypersil BDS C18 (250 mm × 10 mm) semipreparative column was used, mobile phase A was water 5‰ formic acid, and liquid B was methanol containing 5‰ formic acid, and gradient elution (A:B from 90:10 to 0:100) was carried out. The column temperature was normal, the flow rate was 3 mL/min, the injection volume was 0.1 mL], and the detection wavelength was 365 nm to obtain fractions E8-2-1-E8-2-5. Fraction E8-2-4 was subjected to Sephadex LH-20 CC with methanol to obtain fractions E8-2-4-1-E8-2-4-3, among them, E8-2-4-3 was purified by silica gel G CC and eluted with petroleum ether/ ethyl acetate/formic acid (80:1:0.08, 60:1:0.06, v/v) to provide compound 5 (7.1 mg). Fraction E9 was separated by Sephadex LH-20 CC with methanol to produce fractions E9-1-to E9-3, and fraction E9-3 was purified by silica gel G CC eluting with petroleum ether/acetone/formic acid (60:1:0.05, 40:1:0.04, 30:1:0.03, v/v) to obtain compound 6 (4.1 mg). Fraction E9-3-2 was subjected to silica gel G CC eluting with petroleum ether/ acetone/formic acid (50:1:0.05, 40:1:0.04, 30:1:0.03, 20:1:0.02, 10:1:0.01, v/v) to obtain fractions E9-3-2-1-E9-3-2-3, among them, E9-3-2-2 was separated by preparative liquid chromatography to obtain compound 1 (1.2 mg). Fraction E9-2 was purified by preparative liquid chromatography to obtain compounds 7 (136.0 mg) and 8 (5.5 mg). Fraction E10 was isolated by Sephadex LH-20 CC with methanol to obtain fractions E10-1-to E10-5, in which fraction E10-5 was further purified by silica gel G CC and eluted with chloroform/acetone (80:1, 60:1, 50:1, v/v) to provide compound 9 (16.1 mg). Fraction E11 was subjected to Sephadex LH-20 CC with methanol to obtain fractions E11-1-E11-4. Fraction E11-1 was isolated by silica gel G CC eluting with petroleum ether/acetone (70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 0:1, v/v) to obtain fractions E11-1-1-to E11-1-7. Fraction E11-1-5 was subjected to Sephadex LH-20 CC with methanol to obtain compound 11 (15.4 mg) . Fraction E11-1-6 was separated by preparative liquid chromatography to produce compound 12 (2.0 mg). Fraction E11-1-7 was subjected to Sephadex LH-20 CC with methanol twice to obtain compound 13 (1.5 mg). Fractions E11-3 and E11-4 were further separated with preparative liquid chromatography to afford compounds 10 (2.0 mg) and 14 (6.6 mg), respectively. Fraction E13 was separated by a Sephadex LH-20 CC with methanol to obtain fractions E13-1-E13-10, in which component E13-1 was purified on a silica gel G CC and eluted with chloroform/methanol (100:1, 80:1, 75:1, 70:1, 65:1, 60:1, 56:1, 52:1, 50:1, v/v) to produce compound 15 (3.0 mg). Fraction E13-6 was subjected to silica gel G CC and eluted with chloroform/ acetone (25:1, 20:1, 18:1, 16:1, 14:1, v/v) to obtain compound 16 ( Table 1 The NMR data of compound 1 and tryptophan in CD 3 OD a The data were cited from reference [28] (link) Compound 1
Tryptophan a
The extract was subjected to silica gel G column chromatography (CC) and eluted with petroleum ether/ ethyl acetate (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 0:1, v/v), ethyl acetate/methanol (60:1, 40:1, 20:1, 10:1, 4:1, 1:1, 0:1, v/v) and pure methanol in turn to obtain 21 components, E1-E21. Fraction E2 was subjected to Sephadex LH-20 CC with acetone to obtain three fractions, E2-1-to E2-3. Fraction E2-3 was subjected to silica gel G CC eluted with petroleum ether/acetone/ formic acid (1000:10:1, 800:10:0.8, v/v) to obtain compound 2 (4.7 mg). Fraction E4 was subjected to Sephadex LH-20 CC with methanol gel to obtain fractions E4-1-to E4-7. Fraction E4-5 was isolated through silica gel G CC eluted with petroleum ether/acetone (100:1, 80:1, v/v) to obtain compound 3 (4.2 mg). Fraction E6 was separated by Sephadex LH-20 CC with methanol to obtain fractions E6-1-to E6-3, and fraction E6-2 was subjected to silica gel G CC eluted with petroleum ether/ethyl acetate (100:1, 90:1, 80:1, 70:1, 60:1, v/v) to afford compound 4 (3.3 mg). Fraction E8 was separated by preparative liquid chromatography [Hypersil BDS C18 (250 mm × 10 mm) semipreparative column was used, mobile phase A was water 5‰ formic acid, and liquid B was methanol containing 5‰ formic acid, and gradient elution (A:B from 90:10 to 0:100) was carried out. The column temperature was normal, the flow rate was 3 mL/min, the injection volume was 0.1 mL], and the detection wavelength was 365 nm to obtain fractions E8-2-1-E8-2-5. Fraction E8-2-4 was subjected to Sephadex LH-20 CC with methanol to obtain fractions E8-2-4-1-E8-2-4-3, among them, E8-2-4-3 was purified by silica gel G CC and eluted with petroleum ether/ ethyl acetate/formic acid (80:1:0.08, 60:1:0.06, v/v) to provide compound 5 (7.1 mg). Fraction E9 was separated by Sephadex LH-20 CC with methanol to produce fractions E9-1-to E9-3, and fraction E9-3 was purified by silica gel G CC eluting with petroleum ether/acetone/formic acid (60:1:0.05, 40:1:0.04, 30:1:0.03, v/v) to obtain compound 6 (4.1 mg). Fraction E9-3-2 was subjected to silica gel G CC eluting with petroleum ether/ acetone/formic acid (50:1:0.05, 40:1:0.04, 30:1:0.03, 20:1:0.02, 10:1:0.01, v/v) to obtain fractions E9-3-2-1-E9-3-2-3, among them, E9-3-2-2 was separated by preparative liquid chromatography to obtain compound 1 (1.2 mg). Fraction E9-2 was purified by preparative liquid chromatography to obtain compounds 7 (136.0 mg) and 8 (5.5 mg). Fraction E10 was isolated by Sephadex LH-20 CC with methanol to obtain fractions E10-1-to E10-5, in which fraction E10-5 was further purified by silica gel G CC and eluted with chloroform/acetone (80:1, 60:1, 50:1, v/v) to provide compound 9 (16.1 mg). Fraction E11 was subjected to Sephadex LH-20 CC with methanol to obtain fractions E11-1-E11-4. Fraction E11-1 was isolated by silica gel G CC eluting with petroleum ether/acetone (70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 0:1, v/v) to obtain fractions E11-1-1-to E11-1-7. Fraction E11-1-5 was subjected to Sephadex LH-20 CC with methanol to obtain compound 11 (15.4 mg) . Fraction E11-1-6 was separated by preparative liquid chromatography to produce compound 12 (2.0 mg). Fraction E11-1-7 was subjected to Sephadex LH-20 CC with methanol twice to obtain compound 13 (1.5 mg). Fractions E11-3 and E11-4 were further separated with preparative liquid chromatography to afford compounds 10 (2.0 mg) and 14 (6.6 mg), respectively. Fraction E13 was separated by a Sephadex LH-20 CC with methanol to obtain fractions E13-1-E13-10, in which component E13-1 was purified on a silica gel G CC and eluted with chloroform/methanol (100:1, 80:1, 75:1, 70:1, 65:1, 60:1, 56:1, 52:1, 50:1, v/v) to produce compound 15 (3.0 mg). Fraction E13-6 was subjected to silica gel G CC and eluted with chloroform/ acetone (25:1, 20:1, 18:1, 16:1, 14:1, v/v) to obtain compound 16 ( Table 1 The NMR data of compound 1 and tryptophan in CD 3 OD a The data were cited from reference [28] (link) Compound 1
Tryptophan a
The culture supernatants containing the secreted proteins were centrifugated (1 h, 30,000 × g, 10°C) and sterilized by filtration (0.22 µm; Express Plus; Merck Millipore). The supernatants were equilibrated to pH 7 with 0.5 mM NaOH, and the proteins were purified using two ion exchange chromatographies (Fig. S1): first, an anion exchange on DEAE Sephadex A-25 (Cytiva) and a cation exchange on CM Sephadex A-25 (Cytiva) for proteins not retained on the anion exchanger. Both resins were initially equilibrated with 20 mM sodium phosphate buffer (pH 7). Elution of the secreted proteins was performed with 1 M NaCl. Fractions from both anion and cation exchangers were pooled, desalted on Sephadex G-25 (PD-10 Desalting Columns, Cytiva) in 50 mM sodium acetate (pH 5.2), and concentrated using Vivaspin polyethersulfone membrane (3 kDa cutoff, Sartorius). Protein concentrations were determined using the Bradford method (Bio-Rad), and the secretomes were used immediately. Secreted proteins (50 µg) were incubated 30 min with 1 mM hydroxylamine, desalted on Sephadex G-25 (PD SpinTrap, Cytiva), then incubated with 100 mM of H218O2 (Sigma-Aldrich) for 1 h in the dark, then desalted. Initial concentration of commercial H218O2 was estimated to be ∼450 mM using FOX method (51 ). Protein concentrations were determined, and the samples were stored at −20°C.
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More about "Sephadex G 100"
Sephadex G 100 is a crucial tool in the world of molecular biology, biotechnology, and biochemistry.
This size-exclusion chromatography medium is widely used for the separation and purification of a variety of biomolecules, including proteins, nucleic acids, and other macromolecules.
Sephadex G 100 is composed of cross-linked dextran beads, which provide a fractionation range suitable for the analysis of molecules with a molecular weight between 4,000 and 150,000 Daltons.
This versatility makes it a valuable asset in numerous applications, such as protein purification, enzyme studies, and the characterization of protein-protein interactions.
Beyond Sephadex G 100, other related chromatography media like Sephadex® G-25, Sephadex G-50, and Sephadex LH-20 are also commonly employed in various biochemical and biopharmaceutical processes.
These materials offer different fractionation ranges and are often used in combination with Sephadex G 100 to achieve optimal separation and purification.
Additives like Triton X-100 and DMSO can also be utilized in conjunction with Sephadex G 100 to enhance the performance and stability of the chromatography system.
Furthermore, the use of Bovine serum albumin (BSA) as a blocking agent can help to prevent non-specific binding and improve the overall efficiency of the purification process.
Researchers in the field can leverage the power of AI-driven platforms like PubCompare.ai to optimize their Sephadex G 100 protocols.
These platforms offer the ability to easily locate the best protocols from literature, pre-prints, and patents, while providing detailed comparisons to enhance research reproducibility and accuracy.
By embracing these innovative tools, scientists can experience the future of scientific research and unlock new possibilities in their work with Sephadex G 100 and related biomolecular separation techniques.
This size-exclusion chromatography medium is widely used for the separation and purification of a variety of biomolecules, including proteins, nucleic acids, and other macromolecules.
Sephadex G 100 is composed of cross-linked dextran beads, which provide a fractionation range suitable for the analysis of molecules with a molecular weight between 4,000 and 150,000 Daltons.
This versatility makes it a valuable asset in numerous applications, such as protein purification, enzyme studies, and the characterization of protein-protein interactions.
Beyond Sephadex G 100, other related chromatography media like Sephadex® G-25, Sephadex G-50, and Sephadex LH-20 are also commonly employed in various biochemical and biopharmaceutical processes.
These materials offer different fractionation ranges and are often used in combination with Sephadex G 100 to achieve optimal separation and purification.
Additives like Triton X-100 and DMSO can also be utilized in conjunction with Sephadex G 100 to enhance the performance and stability of the chromatography system.
Furthermore, the use of Bovine serum albumin (BSA) as a blocking agent can help to prevent non-specific binding and improve the overall efficiency of the purification process.
Researchers in the field can leverage the power of AI-driven platforms like PubCompare.ai to optimize their Sephadex G 100 protocols.
These platforms offer the ability to easily locate the best protocols from literature, pre-prints, and patents, while providing detailed comparisons to enhance research reproducibility and accuracy.
By embracing these innovative tools, scientists can experience the future of scientific research and unlock new possibilities in their work with Sephadex G 100 and related biomolecular separation techniques.