The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive

2016 ◽  
Vol 120 (11) ◽  
pp. 2878-2885 ◽  
Author(s):  
Kapil Dave ◽  
Hannah Gelman ◽  
Chu Thi Hien Thu ◽  
Drishti Guin ◽  
Martin Gruebele
Keyword(s):  
Fluorescent Protein ◽  
Phosphoglycerate Kinase ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Valentin Dunsing ◽  
Madlen Luckner ◽  
Boris Zühlke ◽  
Roberto A. Petazzi ◽  
Andreas Herrmann ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Living Cells ◽  
Protein Oligomerization ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

2017 ◽  
Author(s):  
Stephen D. Carter ◽  
Shrawan K. Mageswaran ◽  
Zachary J. Farino ◽  
João I. Mamede ◽  
Catherine M. Oikonomou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Low Contrast ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

scholarly journals Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study

2010 ◽  
Vol 1797 (9) ◽  
pp. 1681-1686 ◽  
Author(s):  
Mariam Birungi ◽  
Mihaela Folea ◽  
Natalia Battchikova ◽  
Min Xu ◽  
Hualing Mi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Novel Phosphoprotein-Interacting Region in Nipah Virus Nucleocapsid Protein and Its Involvement in Viral Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9793-9799 ◽  
Author(s):  
Mio Omi-Furutani ◽  
Misako Yoneda ◽  
Kentaro Fujita ◽  
Fusako Ikeda ◽  
Chieko Kai
Keyword(s):  
Amino Acid ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Nipah Virus ◽  
Live Cells ◽  
N Protein ◽  
P Protein ◽  
P Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

ABSTRACT The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.

Expression and localization of atypical PKC isoforms in liver parenchymal cells

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Claudia Stross ◽  
Verena Keitel ◽  
Elisabeth Winands ◽  
Dieter Häussinger ◽  
Ralf Kubitz
Keyword(s):  
Fluorescent Protein ◽  
Hek293 Cells ◽  
Canalicular Membrane ◽  
Liver Parenchymal ◽  
Atypical Pkc ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Parenchymal Cells ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Abstract Members of all three classes of the protein kinase C (PKC) family including atypical PKCzeta (PKCζ) are involved in central functions of liver parenchymal cells. However, expression and localization of PKCiota (PKCι), the highly homologous atypical PKC (aPKC) isoform, in hepatocytes is unknown to date. PKCζ and PKCι were cloned from human and rat liver and fused to fluorescent protein tags (YFP). The sequence of full-length rat PKCι is not yet known and was cloned from cDNA of hepatocytes by the use of degenerated primers. PKCζ-YFP and PKCι-YFP (human and rat) were expressed in HeLa or HEK293 cells and used to test the specificity of seven aPKC antibodies. Two antibodies were PKCι-specific and two were specific for PKCζ in immunofluorescence and Western blot analysis. Subcellular localization was analyzed by immunofluorescence in isolated rat and human hepatocytes and liver sections. Low immunoreactivity for aPKCs was found at the sinusoidal membrane and in the cytosol. The highest density of PKCι as well as PKCζ was found at the canalicular membrane in co-localization with ABC-transporters, such as bile salt export pump or multidrug resistance-associated protein 2. This topology suggests a specific function of aPKCs at the canalicular membrane in addition to their known role in cell polarity of epithelial cells.

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