Sephadex g 25 medium

Manufactured by Merck Group

Sourced in United States

Sephadex G-25 Medium is a size exclusion chromatography resin developed by Merck Group. It is composed of cross-linked dextran beads and is used for the separation and purification of molecules based on their size and molecular weight.

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Lab products found in correlation

Imidazole, by Merck Group (2 mentions) 1 2 dipalmitoyl sn glycero 3 phosphocholine dppc, by Avanti Polar Lipids (1 mentions) Methanol, by Merck Group (1 mentions) Chloroform, by Merck Group (1 mentions) Tris hydroxymethyl aminomethane, by Merck Group (1 mentions) Acetic acid, by Merck Group (1 mentions) L ascorbic acid, by Merck Group (1 mentions) Gvwp filters, by Merck Group (1 mentions) 5 6 carboxyfluorescein cf, by Merck Group (1 mentions)

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Sephadex G-25 (87 citations)

3 protocols using sephadex g 25 medium

Lipid Vesicle Characterization by Fluorescent Probes

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1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) [Avanti Polar Lipids (Alabaster, USA)]; lauric acid (LAH), l-ascorbic acid, tris (hydroxymethyl) aminomethane (Tris), polyoxyethylene-9-lauryl ether (polydocanol), Sephadex G-25 medium, acetic acid, chloroform, and methanol [Sigma Aldrich (St. Louis, USA)] were used as received. 5(6)-Carboxyfluorescein (CF) [Sigma Aldrich] was purified and stocked as the sodium salt [13 ]. The probes 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT1) and tetrasodium 1,3,6,8-pyrenetetrasulfonate (PTS) [Molecular Probes (Eugene, USA)] were used as received.
For LC-MS/MS analysis, acetonitrile (LiChrosolv LC-MS grade) was from Merck (Darmstadt, Germany) and water was from purification system (resistivity > 18 MΩ cm, Sartorius, Göttingen, Germany). Mobile phase components were filtered through 0.45 μm Millipore (Billerica, MA) HVHP and 0.22 μm Millipore GVWP filters, respectively, and degassed in an ultrasonic bath for 15 min.

Farkuh L., Hennies P.T., Nunes C., Reis S., Barreiros L., Segundo M.A., Oseliero Filho P.L., Oliveira C.L., Cassago A., Portugal R.V., Muramoto R.A., Carretero G.P., Schreier S., Chaimovich H, & Cuccovia I.M. (2019). Characterization of phospholipid vesicles containing lauric acid: physicochemical basis for process and product development. Heliyon, 5(10), e02648.

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Recombinant LAPTc Protein Production

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Recombinant LAPTc production was described in [32 (link)]. Briefly, a LAPTc construct was designed, codon optimized for expression in Escherichia coli, synthesized and cloned into the vector pET-19b (Eurofins Genomics, Germany). Production of recombinant LAPTc in E. coli BL21(DE3)pLysS was induced for 20 h at 25°C with 1 mM IPTG and yielded soluble and active enzyme. The protein was purified in two steps by Immobilized Metal Cation Affinity Chromatography (IMAC) and Gel Filtration. For IMAC the nickel matrix was equilibrated with five column volumes (CV) of cold binding buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl). After loading 10 mL of the protein extract, the column was washed with the same buffer until the absorbance at 280 nm stabilized at the baseline. Next, the column was washed with 5 CV of cold washing buffer [50 mM Tris–HCl pH 8.0, 300 mM NaCl, 50 mM imidazole (Sigma, USA)]. Finally, the protein was eluted with 5 CV of cold elution buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl, 400 mM imidazole). The eluates were desalted by gel filtration chromatography, using a NAP-10 column (Sephadex G-25 Medium; Sigma, EUA) to eliminate the imidazole.

Izquierdo M., Lin D., O’Neill S., Webster L.A., Paterson C., Thomas J., Aguado M.E., Colina Araújo E., Alpízar-Pedraza D., Joji H., MacLean L., Hope A., Gray D.W., Zoltner M., Field M.C., González-Bacerio J, & De Rycker M. (2024). Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity. PLOS Neglected Tropical Diseases, 18(2), e0011956.

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Purification of recombinant aminopeptidase enzyme

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Purification of the rLAPLm enzyme was performed by IMAC from E. coli Lemo21(DE3) soluble extracts enriched in the recombinant enzyme, using a 5-mLcolumn packed with a HisPur TM Cobalt matrix (Thermo Scientific/Pierce Biotechnology, USA) in Akta Prime. The matrix was equilibrated with five column volumes (CV) of cold binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl). After loading 100 mL of the protein extract, the column was washed with the same buffer until the absorbance at 280 nm descended until the base-line. Afterward, it was washed with five CV of cold washing buffer [50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole (Sigma, USA)]. Finally, the protein was eluted with a linear gradient of cold elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20-400 mM imidazole). Two mL fractions were collected. Runs were monitored by checking the absorbance at 280 nm, using as blank the corresponding buffer of each step to eliminate the contribution of the imidazole to the absorbance.
The obtained fractions were evaluated by SDS-PAGE. The eluates were desalted by gel filtration chromatography, using a NAP-10 column (Sephadex G-25 Medium; Sigma, USA) to eliminate the imidazole. Afterward, the aminopeptidase EA was assessed toward the Leu-pNA substrate.

Aguado M.E., González-Matos M., Izquierdo M., Quintana J., Field M.C, & González-Bacerio J. (2021). Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase. Protein expression and purification, 183.

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