title: RFP tags for labeling secretory pathway proteins.
abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.
authors.affiliation: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Liyang
authors.initials: L
authors.lastname: Han
authors.affiliation: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Peng
authors.affiliation: Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: pyxu@ibp.ac.cn.
authors.forename: Pingyong
authors.initials: P
authors.lastname: Xu
authors.affiliation: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. Electronic address: shuangyanhuan@163.com.
authors.forename: Shuangyan
authors.initials: S
authors.lastname: Huan
authors.affiliation: Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: mingshu1984@gmail.com.
authors.forename: Mingshu
authors.initials: M
authors.lastname: Zhang
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055571:Secretory Pathway; D013194:Staining and Labeling / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
keywords: Artificial puncta; Evaluation method; K(d); Red fluorescent proteins; pKa