New England Biolabs View Institution's Website 4 articles published in JoVE Biology In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection Bhairavi Jani1, Ryan Fuchs1 1RNA Biology, New England Biolabs This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Biology Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases Paula E. Magnelli1, Alicia M. Bielik1, Ellen P. Guthrie1 1New England Biolabs Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents. Neuroscience High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue Theodore Davis1, Romualdas Vaisvila1 1Applications and Product Development, New England Biolabs The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing β-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site. Biology Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging Christopher R. Provost1, Luo Sun1 1Division of Chemical Biology, New England Biolabs SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.
