Summary

Proteins are distributed in different parts of cells. Subcellular localization analysis of proteins can help to preliminary judgement of protein function. There are two methods at present. One is software prediction, the other is experimental verification.

Software Prediction Method

There are many software for subcellular location prediction. We recommend using Cell-PLoc, a prediction software published on Nature Science by Professor Zhou Guohua, founder of Gordon Life Science Institute.

Cell-PLoc: A package of web-servers for predicting subcellular localization of proteins in various organisms.

software Link:http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/

experimental verification

By marking the target protein, such as fusion green fluorescent protein (GFP), scanning confocal microscopy (SCM) was used to stimulate fluorescence, thus confirming the expression and distribution of the protein. According to the different accuracy of subcellular distribution, the experimental design of subcellular localization can also be divided into simplified subcellular localization analysis and endoplasmic reticulum marker co-localization analysis. The main overexpression plasmid Zoonbio currently uses is pCAMBIA series, which contains one-element and two-element expression vectors. Based on the promoter of Cauliflower mosaic virus 35S (CaMV35S), the target gene is integrated into plants using T-DNA system based on Ti plasmid of Agrobacterium rhizobii, carrying Kana resistance and hygromycin screening markers on the vector. Genome, thus realizing the overexpression of target genes.

For subcellular localization analysis of plant proteins, the localization and co-localization of the fusion fluorescent protein were determined by constructing the over-expression plasmid and transforming the transient expression system of tobacco and protoplast.

Zoonbio can provide scientists with convenient subcellular localization technology. The constructed plasmid was transformed into Agrobacterium tumefaciens strain LBA4404. Tobacco was infected by Agrobacterium tumefaciens mediated method. The positive control was pCAMBIA1302 empty-load. Zeiss, LSM510 Meta, Carl Zeiss AG were used for observation and photography.