Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.     

Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry

Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The technology is especially appropriate for profiling steady-state and dynamic phosphorylation on a proteome-wide scale, as demonstrated through detection of the native phosphorylation of cardiac mitochondrial phosphoproteins and changes in this profile arising from the activity of a protein kinase. For example, Pro-Q Diamond phosphoprotein gel stain was employed to demonstrate that among the 46 subunits of the mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase (complex I), a 42 kDa subunit is phosphorylated in the steady-state. However, exposure of mitochondria to cAMP-dependent protein kinase increases phosphorylation of this 42 kDa subunit and results in de novo phosphorylation of an 18 kDa subunit as well. Since Pro-Q Diamond dye binds to phosphorylated residues noncovalently, the staining technology is fully compatible with modern microchemical analysis procedures, such as peptide mass profiling by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and post-source decay analysis of peptide phosphorylation.     

Reductive PET-fragmentation-cyclization processes of bicyclo[n.3.0]alkanones: synthesis of angular quasi-triquinane and propellane systems

Bicyclo[3.3.0]octanone and bicyclo[4.3.0]nonanone derivatives with a cyclopropane unit in the α-position and an unsaturated side chain in the γ-position of the carbonyl group undergo fragmentation-cyclization processes leading to quasi-triquinane systems upon reductive photoinduced electron transfer (PET). For example, the new angular triquinane derivative 6 and the new propellane derivative 12 were synthesized in one step from these starting materials in moderate to good yields.     

Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.     

En route to coordination chemistry under confined conditions in a porous capsule: Pr3+ with different coordination shells

The highly charged nanocontainer capsule of the type [pentagon]12[linker]30 identical with [(Mo)Mo5O21(H2O)6]12[Mo2O4(SO4)]30 with 20 nanosized pores and channels allows the entrance of cations like Pr3+: the latter are positioned at two different sites and have two different coordination shells, corresponding to a coordination chemistry under confined conditions; a fascinating aspect is that capsule encapsulated water shells fixed by hydrogen bonds act formally as polydentate ligands.     

"Open and Shut" for Guests in Molybdenum-Oxide-Based Giant Spheres, Baskets, and Rings Containing the Pentagon as a Common Structural Element

A novel exchange between ligands and/or guest molecules can be accomplished in giant molecular spheres (an example is shown in the picture) which are in equilibrium with the corresponding giant baskets in solution.