Antarctic krill (E. superba) powder was kindly provided by Zhejiang Hailisheng Biotechnology Co. Ltd. (Zhoushan, China). Trypsin, papain, trifluoroacetic acid, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), phosphate buffered saline, Sephadex G-25, and pepsin, were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Neutrase was purchased from Imperial Jade Biotechnology, Co. Ltd. (Yinchuan, China). Alcalase was purchased from Novozymes Biotechnology Co., Ltd. (Tianjin, China). Diethylaminoethyl (DEAE)-52 cellulose anion exchange resin was purchased from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). Acetonitrile was bought from Thermo Fisher Scientific (Shanghai) Co., Ltd. (Shanghai, China).
Sephadex g 25
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, France
Sephadex G-25 is a cross-linked dextran gel filtration material used for desalting and buffer exchange in laboratory applications. It is designed to separate molecules based on their size, allowing the separation of small molecules from larger ones. The core function of Sephadex G-25 is to facilitate the removal of small molecules, such as salts, from protein or other macromolecule-containing solutions.
Automatically generated - may contain errors
Lab products found in correlation
Acetonitrile, by Merck Group (6 mentions)
Cholesterol, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Methanol, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Pepsin, by Merck Group (2 mentions)
Alcalase, by Novozymes (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Agarose, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Ap132, by Merck Group (2 mentions)
Hydrogen peroxide, by Merck Group (2 mentions)
Triethylamine, by Merck Group (2 mentions)
87 protocols using sephadex g 25
1
Extraction and Characterization of Antarctic Krill Bioactive Compounds
2
Site-specific labeling of Get3 and Mini-Get1/2 proteins
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Get3 with a ybbR tag (DSLEFIASKLA) inserted between residues S110 and D111 was labeled with BDP-maleimide (Thermo Fisher Scientific) or with a 1:1 mixture of ATTO550-maleimide (GE Healthcare) and ATTO 647N-maleimide (ATTO-TEC) via Sfp-catalyzed incorporation of dye-CoA conjugates (Rao et al., 2016 (link); Chio et al., 2019 (link), 2017b (link)). The C-terminal His6-tagged Sfp was removed through Ni-NTA affinity chromatography. The excess dye–CoA conjugates were further removed through gel filtration using a Sephadex G-25 (Sigma-Aldrich) column. Get3ATTO550/ATTO647N-TA complex and Get3BDP-TACm were generated by in vitro translation of TA substrate 3xStrep-SUMOnc-Bos1-opsin in E. coli S30 cell extract (Rao et al., 2016 (link)) supplemented with Get3 variants and untagged T7 RNA polymerase and/or untagged CmRS (for generating Get3BDP-TACm). The resulting Get3-TA complex was purified using Strep-Tactin Sepharose (IBA Life Sciences; Rao et al., 2016 (link); Chio et al., 2019 (link)).
Mini-Get1/2 was labeled with TMR-5-maleimide (Invitrogen) via thiol-maleimide reaction on an engineered single cysteine at residue S77 of Get1 or residue T34 of Get2. The labeling reaction was quenched with 1 mM DTT, and excess dye was removed using a PD-10 column packed with Sephadex G-25 resin (GE Healthcare).
Mini-Get1/2 was labeled with TMR-5-maleimide (Invitrogen) via thiol-maleimide reaction on an engineered single cysteine at residue S77 of Get1 or residue T34 of Get2. The labeling reaction was quenched with 1 mM DTT, and excess dye was removed using a PD-10 column packed with Sephadex G-25 resin (GE Healthcare).
3
Labeling cytoDATL with Cy3 and FRET dyes
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For PIFE, cytoDATL containing an engineered cysteine at G343C was desalted by centrifuging through a 5-ml column (Thermo Fisher Scientific) containing 4 ml bed volume of Sephadex G-25 (Sigma-Aldrich) preequilibrated with labeling buffer (25 mM Tris, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 1 mM imidazole, and 500 µM TCEP). Cy3 maleimide (GE Healthcare, Thermo Fisher Scientific) was added at a 1:1 protein/dye molar ratio. The reaction was incubated for 2 h at RT before being spun at 100,000 rpm (TLA100 rotor; Beckman Coulter) for 15 min at 4°C to remove any precipitate. Labeled cytoDATL was then desalted twice through a column preequilibrated with SEC buffer (25 mM Tris, pH 7.0, 100 mM NaCl, 5 mM MgCl2, and 2 mM EGTA) to remove free Cy3. Typical labeling efficiencies were 20–30%. Cy3 labeling for PIFE in Fig. 1 (B, C, and E) proceeded with the following exceptions: a 1:2 protein/dye ratio in SEC buffer was used, and only a single desalting step followed labeling. For head-to-head FRET, labeling with Alexa Fluor 488 C5 maleimide and Alexa Fluor 647 C2 maleimide (Thermo Fisher Scientific) proceeded with two desalting steps, except the S270C construct for FRET assays was used in place of the G343C construct, and the incubation time with dye was reduced to 30 min. Typical labeling efficiencies were 50–70%.
4
Extraction and Characterization of Medicinal Plant Pectins
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DEAE-cellulose, Sepharose CL-6B, Sephadex G25, and aminobenzamide (2AB) were purchased from Sigma-Aldrich. Endo-polygalacturonase (Endo-PG, EC 3.2.1.15 from Aspergillus niger) was purchased from Megazyme. All chemicals used were analytical grade and produced in China.
Homogalacturonan pectins were extracted from the following plants: Panax japonicus, Pseudostellaria heterophylla, Schisandra chinensis, Prunella vulgaris, Panax Notoginseng, Polygonum orientale, Anemarrhena asphodeloides, Kadsura longipedunculata, Isatis indigotica, Aconitum carmichaelii, Coptis chinensis, and Sophora flavescens.
Homogalacturonan pectins were extracted from the following plants: Panax japonicus, Pseudostellaria heterophylla, Schisandra chinensis, Prunella vulgaris, Panax Notoginseng, Polygonum orientale, Anemarrhena asphodeloides, Kadsura longipedunculata, Isatis indigotica, Aconitum carmichaelii, Coptis chinensis, and Sophora flavescens.
5
Purification and Antiproliferative Activity
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The MCLO-A-3-3 solution (3 mL, 14.1 mg mL−1) was fractionated on a Sephadex G-25 (Sigma-Aldrich, Shanghai, China) column (2.0 × 140 cm) at a flow rate of 1.0 mL min−1. Each eluate (15 mL) was collected and monitored at 280 nm, and four fractions (MCLO-A-3-3-1 to MCLOA-3-3-4) were collected and freeze-dried, and then their antiproliferative activity against the NSCLC cell lines was determined. The fraction having the strongest antiproliferative activity was collected and used for reversed phase-high performance liquid chromatography (RP-HPLC).
6
Glycan-Binding Assay Protocol
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All solvents and reagents
were purchased from commercial sources and were used as received,
unless otherwise noted. Deionized water was used as a solvent in all
procedures. 3-Aminophenylboronic acid, Alizarin Red S (ARS), BSA, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide,
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
and maleimide-functionalized silica beads and Sephadex G-25 were purchased
from Sigma-Aldrich (St. Louis, MO). Glucose, methyl β-O-glucopyranoside, galactose, methyl β-O-galactopyranoside, mannose, methyl α-O-mannopyranoside,
sialic acid, and lactose were purchased from Sigma-Aldrich (St. Louis,
MO). MAA-FITC and SNA-FITC were purchased from Bio-World (Dublin,
OH). 2-α-O-Methyl glycoside of Neu5Ac was synthesized
by a literature method.33
were purchased from commercial sources and were used as received,
unless otherwise noted. Deionized water was used as a solvent in all
procedures. 3-Aminophenylboronic acid, Alizarin Red S (ARS), BSA, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide,
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
and maleimide-functionalized silica beads and Sephadex G-25 were purchased
from Sigma-Aldrich (St. Louis, MO). Glucose, methyl β-O-glucopyranoside, galactose, methyl β-O-galactopyranoside, mannose, methyl α-O-mannopyranoside,
sialic acid, and lactose were purchased from Sigma-Aldrich (St. Louis,
MO). MAA-FITC and SNA-FITC were purchased from Bio-World (Dublin,
OH). 2-α-O-Methyl glycoside of Neu5Ac was synthesized
by a literature method.33
7
Ganglioside Extraction from Brain Tissue
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Ganglioside extraction procedure was performed as previously described [31 (link),69 (link),70 (link)]. Briefly, dissected brain regions were homogenized in ice-cold distilled water (W) in a Potter–Elvehjem glass-Teflon homogenizer (DeOtto Lab, Zagreb, Croatia). Lipids were extracted using organic solvents chloroform (C):methanol (M) (1:2, by vol.), followed by partition and repartition by adding M and W to a final volume ratio 1:1:0.7 (chloroform was from T.T.T., Sveta Nedjelja, Croatia; methanol from Honeywell Riedel-de Haen, Seelze, Germany). Upper phases were collected, evaporated to dryness and further purified by gel filtration Sephadex-G25 (Sigma-Aldrich, St. Louis, MO, USA) [71 (link)].
8
Protein Labeling with Fluorescent Dyes
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Dye NHS esters were dissolved in DMSO to a concentration of 2 mg/mL. Secondary antibodies were dissolved/diluted to 2mg/mL in PBS buffer of a pH of 8.3, adjusted with sodium bicarbonate buffer. For antibodies purchased with concentrations lower than 2 mg/mL, proteins were first concentrated with Amicon® Ultra Centrifugal Filters (UFC501096, EMD, Millipore). A 50 μL dye-NHS solution was slowly added to a 0.5 mL protein solution. Reaction was incubated at RT for 60 min under constant but slow stirring. The labeled proteins could be separated from unreacted dye NHS esters by gel permeation chromatography with Sephadex™ G-25 (G25150 SIGMA) with a column of diameter around 1 cm and length longer than 12 cm. Sephadex® G-25 was first swelled in PBS buffer at 90 °C water bath for 1 h. After gel settling down in room temperature, buffer was exchange to fresh PBS buffer and gels were stored at 4 °C. For gel chromatography, column was packed with swelled gels, and then equilibriumed with PBS. The labeled protein solution was then loaded and eluted with PBS buffer. First band with light color for dye conjugated proteins was collected. This solution was then centrifuged and supernatant was collected and concentrated with Amicon® Ultra Centrifugal Filters (UFC501096, EMD, Millipore) into a final concentration of 1 - 2 mg/mL in PBS with 30% glycerol and 5 mM sodium azide and stored at -20 °C.
9
Synthesis and Labeling of BODIPY-FL-CoA
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BODIPY-FL-CoA was synthesized and purified as described (Yin et al., 2006 (link)) with the exception that BODIPY-FL maleimide (Life Technologies) was used instead of Alexa Fluor 488 C5 maleimide. The lyophilized compound was dissolved in DMSO, and dye concentration was quantified after dilution in methanol using ε504 = 79,000 M−1cm−1.
30 µM ybbR-Get3 was mixed with 60 µM BODIPY-FL-CoA and 12 µM Sfp-His6 in Sfp labeling buffer (50 mM KHEPES, pH 7.4, 10 mM MgCl2) in a total volume of 800 µL. The reaction mixture was rotated at room temperature for 1 hr. 10 µL 2 M imidazole (pH 7.0) was added before passing the reaction through Ni-NTA to remove Sfp-His6. Gel filtration through a Sephadex G-25 (Sigma-Aldrich) column was used to remove excess BODIPY-FL-CoA and exchange ybbR-Get3BDP into GET buffer (50 mM KHEPES (pH 7.5), 150 mM KOAc, 5mM Mg(OAc)2, and 1 mM DTT). Translocation and ATPase reactions mediated by ybbR-Get3BDP were performed as described in Rome et al. (2013) (link).
30 µM ybbR-Get3 was mixed with 60 µM BODIPY-FL-CoA and 12 µM Sfp-His6 in Sfp labeling buffer (50 mM KHEPES, pH 7.4, 10 mM MgCl2) in a total volume of 800 µL. The reaction mixture was rotated at room temperature for 1 hr. 10 µL 2 M imidazole (pH 7.0) was added before passing the reaction through Ni-NTA to remove Sfp-His6. Gel filtration through a Sephadex G-25 (Sigma-Aldrich) column was used to remove excess BODIPY-FL-CoA and exchange ybbR-Get3BDP into GET buffer (50 mM KHEPES (pH 7.5), 150 mM KOAc, 5mM Mg(OAc)2, and 1 mM DTT). Translocation and ATPase reactions mediated by ybbR-Get3BDP were performed as described in Rome et al. (2013) (link).
10
Anti-Aging Compound Fractionation
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The LP-α-II-IV solution (2 mL, 23.5 mg mL−1) was fractionated on a Sephadex G-25 (Sigma-Aldrich, Shanghai, China) column (2.5 × 120 cm) at a flow rate of 2.0 mL min−1. Each eluate (80 mL) was collected and monitored at 280 nm, and four fractions (LP-α-II-IV-I ∼ LP-α-II-IV-IV) were collected and anti-aging activity in N2 nematodes was detected. The fraction having the strongest anti-aging activity was collected and prepared for Reversed Phase-High Performance Liquid Chromatography (RP-HPLC).
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