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Isolation of circulating human monocytes with high purity for proteomic analysis

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Gonzalez Barderas, Maria & Gallego-Delgado, Julio & Mas, Sebastián & Duran, Mari Carmen & Lázaro, Alberto & Hernandez Merida, Sergio & Egido, Jesús & Vivanco, Fernando, Fernando Vivanco .Isolation of circulating human monocytes with high purity for proteomic analysis.

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Gonzalez Barderas, Maria & Gallego-Delgado, Julio & Mas, Sebastián & Duran, Mari Carmen & Lázaro, Alberto & Hernandez Merida, Sergio & Egido, Jesús & Vivanco, Fernando. Isolation of circulating human monocytes with high purity for proteomic analysis.

https://hdl.handle.net/20.500.12080/39714
dc.contributor.advisor Fernando Vivanco
dc.contributor.author Gonzalez Barderas, Maria
dc.contributor.author Gallego-Delgado, Julio
dc.contributor.author Mas, Sebastián
dc.contributor.author Duran, Mari Carmen
dc.contributor.author Lázaro, Alberto
dc.contributor.author Hernandez Merida, Sergio
dc.contributor.author Egido, Jesús
dc.contributor.author Vivanco, Fernando
dc.date.accessioned 2024-02-12T13:45:19Z
dc.date.available 2024-02-12T13:45:19Z
dc.date.created 2004
dc.identifier.uri https://hdl.handle.net/20.500.12080/39714
dc.description.abstract We describe a simple method for isolation of human blood monocytes with the high purity (95¿98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albu min and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to para magnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 107 monocytes with . 96% purity are routinely obtained. From the isolated monocytes 200¿250 mg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3¿10, 4¿7, 6¿9 and 6¿11 pH range. DNA was the main contaminant that interfered with the 2- DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4¿7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4¿7, 6¿9 and 6¿11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient. Keywords: Human monocytes / Isolation method / Proteomic analysis / Two-dimensional gel electrophoresis es_ES
dc.format application/pdf es_ES
dc.language eng es_ES
dc.rights CC-BY es_ES
dc.rights.uri http://creativecommons.org/licenses/by/4.0/deed.es es_ES
dc.title Isolation of circulating human monocytes with high purity for proteomic analysis es_ES
dc.type info:eu-repo/semantics/article es_ES
dc.rights.accessrights info:eu-repo/semantics/restrictedAccess es_ES
dc.identifier.location N/A es_ES


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