Hussain Noor Feuza, Mohammed Sulma Ibrahim
Franklin College, IUPUI- Indiana University Purdue University Indianapolis, Indianapolis, Indiana, USA.
Department of Comparative Pathobiology, Purdue University Center for Cancer Research, Purdue University, Indiana, USA.
J Oncol Res Ther. 2018;4(1). Epub 2018 Feb 20.
Laser Capture Micro-dissection (LCM) is a technique that is used to isolate specific tumor cells from a heterogeneous tumor tissue sample.To aid in identifying and dissecting pure tumor cells from other parts of the tissues such as the stroma, tissues are stained with Haematoxylin and Eosin. The cells are then used for protein, RNA or DNA extraction. However, the effect of Haematoxylin and Eosin or other different stains routinely used in the laboratories on the recovery, quantity and quality of proteins especially for down stream application such as 2-dimenssional gel electrophoresis (2-DE) and MS not known. This study, determined the effect of Haematoxylin staining on the detection methods used in 1-D SDS-PAGE for protein quantification. A series of concentration of proteins were obtained from human pancreatic whole tissue and was run on a SDS-PAGE parallel with the proteins obtained from Haematoxylin stained and unstained tissues.The protein band intensities were measured with a densitometer after separately stained with SYPRO Ruby or CBB R-250.The protein band intensity ratios of the whole tissue and Haematoxylin stain/ Haematoxylin unstained tissue were calculated. According to the ratios,there was an intensity loss in the Haematoxylin stained proteins when detecting through CBB R -250 but not from SYPRO Ruby. This was due to the structure and reactivity of these two stains towards proteins in the presence of Haematoxylin. The study recommends the use of SYPRO Ruby instead of CBB R-250 to visualize proteins in 2-DE gels when tissues were stained with Haematoxylin.
激光捕获显微切割（LCM）是一种用于从异质性肿瘤组织样本中分离特定肿瘤细胞的技术。为了有助于从诸如基质等组织的其他部分识别和切割出纯肿瘤细胞，组织用苏木精和伊红染色。然后将这些细胞用于蛋白质、RNA或DNA提取。然而，实验室常规使用的苏木精和伊红或其他不同染色剂对蛋白质的回收率、数量和质量的影响，尤其是对于二维凝胶电泳（2-DE）和质谱等下游应用的影响尚不清楚。本研究确定了苏木精染色对一维SDS-PAGE中用于蛋白质定量的检测方法的影响。从人胰腺全组织获得一系列蛋白质浓度，并与从苏木精染色和未染色组织获得的蛋白质一起在SDS-PAGE上进行电泳。用SYPRO Ruby或考马斯亮蓝R-250分别染色后，用密度计测量蛋白质条带强度。计算全组织与苏木精染色/未染色苏木精组织的蛋白质条带强度比。根据这些比率，通过考马斯亮蓝R-250检测时，苏木精染色的蛋白质存在强度损失，但用SYPRO Ruby检测时没有。这是由于在苏木精存在下这两种染色剂对蛋白质的结构和反应性不同。该研究建议，当组织用苏木精染色时，在二维凝胶电泳中使用SYPRO Ruby而不是考马斯亮蓝R-250来可视化蛋白质。
