A preparation method of colloidal gold-carbocyanine-low-density lipoprotein double-labeled probe

Abstract

The invention relates to a method for preparing a colloidal gold-carbonylated cyanine-low-density lipoprotein (LDL) double-labeled probe, which is characterized in that two different tracers of colloidal gold and carbonylated cyanine (DiI) are used to label the same target protein Prepare double-labeled probes on the molecule, and use instruments and equipment to detect the functional activity of cell protein receptors. The preparation steps include: (1) extract and purify LDL; (2) prepare DiI-LDL; (3) prepare colloidal gold; (4) prepare Colloidal gold-DiI-LDL double-labeled probe; (5) Application of colloidal gold-DiI-LDL double-labeled probe. By improving the pH of the preparation environment, protein quantification and increasing the number of separations, the prepared colloidal gold-DiI-LDL double-labeled probe has the advantages of high yield, high purity, strong specificity, wide application range and rich detection methods. Laser confocal microscopy, flow cytometry and transmission electron microscopy and other instruments and equipment can accurately and efficiently detect the functional activity of cellular LDL receptors.

Description

1. Technical field [0001] The present invention relates to a method for preparing colloidal gold-carbonylated cyanine (1,1'Dioctadecyl 3,3,3',3'tetramethylindocarbocyanine perchlorate, DiI)-low density lipoprotein (Low Density Lipoprotein, LDL) double-labeled probe, which The characteristic is that two different tracers of fluorescein DiI and colloidal gold are used to successively label the same ligand LDL to prepare double-labeled probes, and the functional activity of cell protein receptors is detected by instruments and equipment. 2. Background technology [0002] Low Density Lipoprotein Receptor (LDL-R) is widely distributed in various cells and tissues of humans and animals, such as liver cells, lymphocytes, monocytes and macrophages, and ovaries. LDL-R is a transmembrane glycoprotein located on the surface of the cell membrane. The endocytosis mediated by it is the main pathway of plasma low-density lipoprotein cholesterol (LDL-C) metabolism. The amount of LDL intake ...

AI Technical Summary

Problems solved by technology

[0007] In order to solve the shortcomings that a single tracer probe cannot use multiple detection methods to analyze the LDL-R functional activity of the same batch of cells at the same time, and it is difficult to observe the distribution and metabolism of LDL in the cells, overcome the colloidal gold-DiI- The LDL preparation method has the disadvantages of low probe yield, poor purity, and inhomogeneous colloidal gold particle size. The present invention utilizes mature LDL lipoprotein extraction and purification, DiI-labeled protein and colloidal gold-labeled protein technologies, combined with previous research experience, first using fluorescence DiI-labeled LDL, on the basis of DiI-LDL, is then labeled with colloidal gold to prepare a double-labeled probe colloidal gold-DiI-LDL. The double-labeled probe of the present invention can be used for LDL of liver cells and macrophages -R functional activity for accurate and efficient analysis

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