The two-step purification of MpBgl3 was performed at 4 °C, in which 100 mL of the crude enzyme extract was concentrated and fractionated by tangential filtration using a Vivaspin™ 20 membrane (50 and 100 kDa cutoff, GE Healthcare Life Sciences, Uppsala, Uppland, SE). In this step the proteins greater than 50 kDa and smaller than 100 kDa were recovered in a total volume of 10 mL. the pH of this recovered fraction was adjusted to 7.0 with 25 mM Tris-HCl buffer pH 7.0, and loaded onto a Fractogel® EMD DEAE(M) (Merck Millipore Corporation, Darmstadt, Hessen, DE) (3 ×1 cm) column previously equilibrated with the same Tris-HCl buffer (25 mM, pH 7.0). The protein was eluted with a linear gradient of 0 to 1 M sodium chloride, and the fractions with Bgl activity were pooled and used in all subsequent experiments. The enzymatic extract and purified MpBgl3 were analyzed by SDS-PAGE 12%, stained with Coomassie Blue, and the protein concentration was estimated by the Bradford method37 (link).
Fractogel emd deae m
Manufactured by Merck Group
Sourced in Germany
Fractogel® EMD DEAE(M) is a chromatography resin used for the purification and separation of biomolecules. It is a diethylaminoethyl (DEAE) anion exchange medium that can be used for the capture and purification of proteins, enzymes, and nucleic acids.
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Lab products found in correlation
2 protocols using fractogel emd deae m
1
Purification of MpBgl3 Enzyme
2
Recombinant Expression and Purification of Om45p
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DNA encoding the soluble domain of Om45p without the first 22 amino acids (predicted N‐terminal transmembrane domain) was cloned onto the expression vector pET15b (Novagen‐Addgene, Cambridge, MA, USA) and expressed in E. coli BL21 (DE3). The expressed chimeric protein containing a His6 tag on its N‐terminus, a thrombin cleavage site, and N‐terminally truncated Om45p was affinity purified with Ni2+ chelating Sepharose (GE Healthcare, Piscataway, NJ, USA), concentrated 10 times with a Millipore Amicon Ultra‐15 10000 NMWL concentrator (Carrigtwohill, Cork, Ireland) in a table centrifuge at 4°C and subjected to anion‐exchange chromatography in Fractogel EMD DEAE (M) (Merck KGaA, Darmstadt, Germany), in a 10 × 150 mm column (20 mM Tris/HCl pH 7.4, linear gradient elution with NaCl from 50 to 200 mM at the flow rate 1 ml/min in 25 min). The purity of the peak fractions was controlled by SDS‐PAGE and mass spectrometry. Peak fractions were concentrated to 1.69 mg of protein per ml. 500 µl of the protein were used for the generation of polyclonal antisera fin two rabbits by Davids Biotechnologie (Regensburg, Germany).
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