Preparation method of phycoerythrin immunofluorescence probe

Abstract

The invention discloses a preparation method of phycoerythrin immunofluorescent probe, which comprises the step of labeling phycoerythrin on a target protein; before the step of labeling phycoerythrin on a target protein, the method It also includes the step of blocking the free sulfhydryl groups on the target protein or phycoerythrin. In the present invention, before labeling and cross-linking phycoerythrin to the target protein using conventional method steps, the free sulfhydryl groups on the target protein or phycoerythrin are first blocked. After the free sulfhydryl groups are blocked, the amine-thiol cross-linking The reagent can only react with the amino group on the blocked target protein or phycoerythrin, but not with the sulfhydryl group on the corresponding protein, so that it can ensure that all the amino groups of the amine-sulfhydryl cross-linking agent are used to couple the sulfhydryl algae Phycoerythrin or target protein, thereby effectively improving the labeling efficiency of phycoerythrin on the target protein, and the obtained phycoerythrin immunofluorescence probe has a high signal-to-noise ratio in immunodetection.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to a method for preparing a phycoerythrin immunofluorescence probe. Background technique [0002] Phycoerythrin (P-phycoerythrin, referred to as PE) is isolated and purified from red algae and is currently a new type of fluorescent labeling reagent that is commonly used. Under the excitation of specific wavelength, phycobiliprotein can emit strong fluorescence, its fluorescence intensity is 30-100 times that of fluorescein, it has good light absorption performance and high quantum yield, and has a wide excitation in the visible spectrum region and launch range. [0003] The use of phycoerythrin in fluorescence analysis has incomparable advantages over traditional chemical fluorescent dyes. For example: (1) It has a broad absorption spectrum in a wide pH range, and it is relatively easy to select a suitable excitation wavelength to obtain efficient fluorescence emiss...

AI Technical Summary

Problems solved by technology

[0008] The disadvantage of the above-mentioned PE labeling method is that: since the target protein and phycoerythrin carry both free amino groups and free sulfhydryl groups, when amine-sulfhydryl cross-linking agents such as SMCC are used for activation, the amine-sulfhydryl cross-linking agents will not only Binding to amino groups also binds to sulfhydryl groups, which leads to the fact that when proteins activated by amine-sulfhydryl crosslinkers are cross-linked with thiolated phycoerythrin, those on proteins activated by amine-sulfhydryl crosslinkers that are already bound to sulfhydryl groups The cross-linking agent is difficult to re-cross-link with thiolated phycoerythrin, resulting in less phycoerythrin bound to the target protein and poor labeling effect. low noise ratio

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