1 st experiment:
293T and 293T cells expressing a deletion form of FBLN7 lacking the coiled-coil domain, were grown to confluence and the resulting serum-free conditioned medium (CM) was harvested every 2 days. The collected CM was concentrated by protein precipitation with 80%-saturated ammonium sulfate and dissolved in 50 mM Tris-HCl (pH 7.4) containing 150 mm NaCl supplemented with 5 mM CaCl2, 0.1% Triton X-100, and a proteinase inhibitor mixture [0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 0.16 M aprotinin, 0.025 mM bestatin, 7.5 M E-64, 0.01 mM leupeptin, and 5 M pepstatin] . The precipitants were dialyzed against a 400 times volume of diluent buffer. The dialyzed 293T CM was passed through a Cobalt-affinity column once to eliminate non-specific bound proteins, and the flow-through fraction was collected and mixed with deltaCC, and then applied to the column chromatography. deltaCC and its affinity-binding proteins bound to the column, and 1200 mM NaCl was used to elute the proteins that were associated with deltaCC. The eluted samples were subjected to SDS-PAGE and separated proteins were visualized by Coomassie staining and excised.
2nd experiment:
CHO cells expressing full-length FBLN7 were grown to confluence and the resulting serum-free conditioned medium (CM) was harvested every 2 days. The collected CM was concentrated by protein precipitation with 80%-saturated ammonium sulfate and dissolved in phosphate-buffered saline without calcium and magnesium [PBS (-)] supplemented with 0.1% Triton X-100 and a proteinase inhibitor mixture [0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 0.16 M aprotinin, 0.025 mM bestatin, 7.5 M E-64, 0.01 mM leupeptin, and 5 M pepstatin]. The precipitants were dialyzed against a 400 times volume of PBS (-) plus Triton X-100 and then applied to a Nickel (Ni)-Sepharose Histidine-affinity column. Bound proteins were eluted with PBS (-) supplemented with 500 mM NaCl, 500 mM imidazole, and 0.01% Triton X-100. To exclude non-specific bound proteins, the fractions containing FBLN7 were applied to a heparin-Sepharose-6B column. FBLN7 bound to the column and mainly eluted at 0.6 M NaCl. This fraction was further purified by Ni-Sepharose column chromatography and the final elute fraction containing FBLN7 and its putative high-affinity binding proteins was subjected to SDS-PAGE. Separated proteins were detected by Coomassie staining and protein bands were excised.
[doi:10.25345/C5G55G]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Skin aging, stem cell aging, epidermal stem cells, slow-cycling cell, extracellular matrix, inflammation, fibulin, lineage tracing, stem cell plasticity, wound healing
Principal Investigators: (in alphabetical order) |
Hiromi Yanagisawa, M.D. Ph.D., University of Tsukuba, Japan |
Submitting User: | mogoodarzi |
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Owner | Reanalyses | |
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