Recombinant human netrin‐1 (R&D) and GDNF (PeproTech) were iodinated with 125I‐Na using the lactoperoxidase method. Proteins were dissolved in 30 µl of 0.25 M phosphate buffer, pH 7.5, and mixed with 125I‐Na (1 mCi 37 mBq; GE Healthcare). The reaction was started by adding lactoperoxidase 10 µl of 50 µg/ml and 0.05% H2O2. The mixture was incubated at room temperature for 20 min and the reaction was stopped by adding 3 vol of 0.1 M phosphate buffer, pH 7.5, containing 0.1 M NaI, 0.42 M NaCl, and 25 µl of 2.5% BSA. Free iodine and iodinated growth factors were separated by Sephadex G‐25 columns (PD10; GE Healthcare). For column equilibrium and elution, 0.1 M phosphate buffer, pH 7.5, with 1% BSA was used. The iodinated proteins were concentrated by using YM‐10 Centricon columns (Millipore). The specific activity of 125I‐labeled netrin‐1 and GDNF was > 108 cpm/µg protein.
Ym 10 centricon columns
Manufactured by Merck Group
The YM-10 Centricon columns are lab equipment designed for concentration and desalting of macromolecules. They utilize a membrane with a molecular weight cutoff of 10 kDa to selectively retain molecules above that size while allowing smaller molecules to pass through.
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Lab products found in correlation
3 protocols using ym 10 centricon columns
1
Iodination of Netrin-1 and GDNF Proteins
2
Purification of GST-tagged MYF5 Proteins
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GST-tagged full-length MYF5 and partial MYF5 domains were cloned in plasmid vector pGEX4T2, and transformed into competent BL21 E. coli. After overnight culture, cells were treated with 0.5 mM IPTG to induce GST-MYF5 production. Cells were then lysed in GST lysis buffer (50 mM Tris-HCl [pH 8.8], 200 mM NaCl, 1 mM EDTA) containing protease inhibitor using sonication and the lysate was cleared by centrifugation. The supernatant was supplemented with Triton X-100 (3%), and then incubated with Glutathione Sepharose 4B beads (GE Healthcare Life Sciences) to allow binding. Following washes with GST lysis buffer containing 1% Triton X-100 and elution with a buffer containing 300 mM Tris-HCl [pH 8.8], 120 mM NaCl and 20 mM Glutathione, GST-tagged proteins were dialyzed using the YM-10 centricon columns (Millipore) and washed with TBS buffer before use in binding assays.
3
Affinity Purification of FLAG-Tagged NRPA2 and NRPC2
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150–250 g of fresh or frozen leaf tissue expressing FLAG-tagged NRPA2 or NRPC2 was ground in extraction buffer (300 mM NaCl, 20 mM Tris pH 7.5, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1:200 plant protease inhibitor cocktail (Sigma)) at 4°C, filtered through two layers of Miracloth (Calbiochem) and centrifuged twice at 7000–10 000 x g, 25 min, 4°C. Supernatants were incubated with anti-FLAG-M2 resin for 3 h and a 15 ml tube using 50 ul of resin per 14 ml of extract. Pooled resin was washed seven times in 14 ml of extraction buffer containing 0.4% Nonidet P-40 (Sigma). Wash buffer was removed from the resin before adding 1 volume of Ag/Ab Elution Buffer (Pierce) at 4°C. The sample was mixed thoroughly and incubated for 3 min on ice. The resin was pelleted, and the supernatant, containing eluted proteins, was recovered in 500 ul aliquots, concentrated using YM-10 Centricon columns (Millipore) at 4°C and desalted twice using a Pierce 500 ul desalting column. Final samples, eluted in 25 mM ammonium bicarbonate, were subjected to LC–MS/MS.
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