Gonzalez-Barderas Maria, Gallego-Delgado Julio, Mas Sebastián, Duran Mari Carmen, Lázaro Alberto, Hernandez-Merida Sergio, Egido Jesús, Vivanco Fernando
Department of Immunology, Fundación Jiménez Diaz, Madrid, Spain.
Proteomics. 2004 Feb;4(2):432-7. doi: 10.1002/pmic.200300629.
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 (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic 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, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg 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.
我们描述了一种简单的方法，用于分离蛋白质组分析所需的高纯度（95 - 98%）人血单核细胞，该方法可避免其他血细胞（血小板和淋巴细胞）以及最丰富的血浆蛋白（白蛋白和免疫球蛋白）的污染。通过梯度离心，然后用偶联至顺磁珠的特异性单克隆抗体进行阳性选择来纯化血单核细胞。对阳性选择步骤的洗脱条件进行了修改，以避免白蛋白污染。该方法与用于评估细胞群体纯度的流式细胞术兼容。从28 mL血液中，通常可获得纯度> 96%的10⁷个单核细胞。从分离的单核细胞中可回收200 - 250 μg蛋白质。整个方法可在三小时内完成。使用阴性选择步骤也获得了类似结果，但纯度较低（92%），成本增加且时间更长。单核细胞溶解后，通过二维凝胶电泳（2 - DE）在3 - 10、4 - 7、6 - 9和6 - 11 pH范围内分析蛋白质。DNA是干扰2 - DE的主要污染物，通过用DNA酶处理将其去除。凝胶图像分析允许在4 - 7 pH范围内可重复检测和定量1500个斑点，并通过合并（重叠）4 - 7、6 - 9和6 - 11 pH范围内的二维凝胶总共检测和定量超过2000个斑点。由于其快速性和可重复性，该方法对于大量患者的单核细胞临床研究很有用，它允许对正常样本与病理样本进行比较分析，并允许对每个患者单核细胞表达的蛋白质进行跟踪。
