二抗孵育(Secondary antibody inucubation) 二抗一般用辣根过氧化物酶(HRP)标记的二抗。 二抗也用5%的脱脂奶稀释,一般按照1:5000加入。将膜放入到含有一抗的自封袋中,用封口器将自封袋封好,用胶带粘到静音混合器上,稀释的一抗一般加入3ml,孵育2h。2h后,将膜拿出,放在含有0.05%吐温的PBST溶液中,在脱色摇床上洗脱...
利用抗原-抗体反应，可以在所有蛋白质中只Detection特定蛋白质。本文的Western Blotting使用神经细胞分化前的...
(Western Blot) 蛋白免疫印迹杂交(Western Blot WB)是将蛋白样本通过聚丙烯酰胺电泳按分子量大小分离,再转移到杂交膜(blot)上,然后通过一抗/二抗复合物对靶蛋白进行特异性检测的方法。WB是进行蛋白质分析最流行和成熟的技术之一。本指南讨论Western Blot操作方法及常见问题分析,有助于成功完成WB。 A 蛋白样本提取制备...
检测指标:An MSD western-replacement method (Meso Scale Discovery) using specific anti-alpha smooth muscle actin (alpha-SMA) antibody was used to quantify SMA in protein lysates. Samples (25 uL) were added to multi-array 96-well plates (high bind, Meso Scale Discovery) and incubated for 2 h...
Western免疫印迹(Western Blot)是将蛋白质转移到膜上,然后利用抗体进行检测。对已知表达蛋白,可用相应抗体作为一抗进行检测,对新基因的表达产物,可通过融合部分的抗体检测。 Western Blot采用的是聚丙烯酰胺凝胶电泳,被检测物是蛋白质,“探针”是...
site of enzymatic activity on the blot. The WesternBreeze kit has been optimized to provide picogram sensitivity. Each kit includes ready-to-use or easy-to-dilute blockers, primary antibody diluent, wash solution, conjugated secondary antibody solution, substrate, and two convenient ...
Western,也称Western blot、Western blotting、Western印迹,是用抗体检测蛋白的重要方法之一。可按照以下步骤操作。 1.收集蛋白样品(Protein sample preparation) 使用细胞裂解液,对贴壁细胞、悬浮细胞或组织样品进行裂解。然后测定每个蛋白样品的蛋白浓度。 2. 电泳(Electrophoresis) ...
antibody binding was prevented by incubation of 150 μL of 3% blocking buffer (0.6 g of MSD Blocker A in 20 mL bidest) for 1 h at room temperature. For the detection of bound alpha-SMA, an anti-alphaSMA antibody (1:5000; Sigma-Aldrich) and goat anti-mouse sulfo-TAG antibody (1:...
2.Burnette W N . "Western Blotting": Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A[J]. Analytical Biochemistry, 1981, 112(2):195-203. ...
Santa Cruz Biotechnology) or horseradish peroxidase–conjugated anti-mouse IgG secondary antibody (for Oatp1b2, Mdr1, and Na1/K1 ATPase; 1:10,000; catalog number 115-035-003; Jackson ImmunoResearch, West Grove, PA) in 5% milk for 1 hour at room temperature. Detection was enhanced using chem...