The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employ...The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest(POI).However,the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags.Conversely,HaloTag,a novel self-labeling enzyme(SLE)tag,could quickly form a covalent bond with its ligand,enabling fast and specific labeling of POI.These desirable features greatly increase the accuracy and specificity,making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI.Notably,the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency.In this review,we summarize the development of HaloTag and recent advanced investigations associated with HaloTag,including in vitro imaging(e.g.,POI imaging,cellular condition monitoring,microorganism imaging,system development),in vivo imaging,biomolecule immobilization(e.g.,POI collection,protein/nuclear acid interaction and protein structure analysis),targeted degradation(e.g.,L-AdPROM),and more.We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.展开更多
Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one o...Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one of the most powerful methods to map protein RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method （GoldCLIP） that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein （PTB）, a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.展开更多
基金The authors are grateful for financial support from the University of Wisconsin—Madison and National Institutes of Health(P30CA014520).
文摘The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest(POI).However,the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags.Conversely,HaloTag,a novel self-labeling enzyme(SLE)tag,could quickly form a covalent bond with its ligand,enabling fast and specific labeling of POI.These desirable features greatly increase the accuracy and specificity,making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI.Notably,the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency.In this review,we summarize the development of HaloTag and recent advanced investigations associated with HaloTag,including in vitro imaging(e.g.,POI imaging,cellular condition monitoring,microorganism imaging,system development),in vivo imaging,biomolecule immobilization(e.g.,POI collection,protein/nuclear acid interaction and protein structure analysis),targeted degradation(e.g.,L-AdPROM),and more.We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.
基金supported in part by grants from the Ministry of Science and Technology of China(Grant No.2017YFA0504200 to YY,Grant Nos.2012CB910502 and 2011CB966304 to JM)the National Natural Science Foundation of China(Grant Nos.91640105 and 31770875 to YY,Grant No.31230041 to JM,and Grant Nos.91640115 and 31670827 to YZ)
文摘Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one of the most powerful methods to map protein RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method （GoldCLIP） that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein （PTB）, a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.
