1.Synthesis, labeling and bioanalytical applications of a tris(2,2'-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.
Zhou X1, Zhu D1, Liao Y1, Liu W1, Liu H1, Ma Z1, Xing D1. Nat Protoc. 2014 May;9(5):1146-59. doi: 10.1038/nprot.2014.060. Epub 2014 Apr 17.
Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)3(2+) probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)3(2+)-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead-based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)3(2+)).
2.The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.
Cihil KM1, Swiatecka-Urban A. J Vis Exp. 2013 Dec 13;(82):e50867. doi: 10.3791/50867.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione.
3.Facile one-step coating approach to magnetic submicron particles with poly(ethylene glycol) coats and abundant accessible carboxyl groups.
Long G1, Yang XL, Zhang Y, Pu J, Liu L, Liu HB, Li YL, Liao F. Int J Nanomedicine. 2013;8:791-807. doi: 10.2147/IJN.S41411. Epub 2013 Feb 25.
PURPOSE: Magnetic submicron particles (MSPs) are pivotal biomaterials for magnetic separations in bioanalyses, but their preparation remains a technical challenge. In this report, a facile one-step coating approach to MSPs suitable for magnetic separations was investigated.
4.Synthesis of photolabile transcription initiators and preparation of photocleavable functional RNA by transcription.
Huang F1, Shi Y. Bioorg Med Chem Lett. 2012 Jul 1;22(13):4254-8. doi: 10.1016/j.bmcl.2012.05.028. Epub 2012 May 17.
Two new photolabile adenosine-containing transcription initiators with terminal thiol and amino functionalities are chemically synthesized. Transcription in the presence of the transcription initiators under the T7 phi2.5 promoter produces 5' thiol- and amino-functionalized RNA conjugated by a photocleavable (PC) linker. Further RNA functionalization with biotin may be achieved through acyl transfer reactions from either biotinyl AMP to the RNA thiol group or biotin NHS to the RNA amino group. Photocleavage of the PC linker displays relatively fast kinetics with a half-life of 4-5 min. The availability of these transcription initiators makes new photolabile RNA accessible for affinity purification of RNA, in vitro selection of functional RNAs, and functional RNA caging.
