Significance of Using SYPRO Ruby against CBB R-250 for Visualizing Haematoxylin Stained Proteins in Gels.

Abstract

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.