Download (.pdf) The statistics of protein expression ratios for cellular fluorescence studies
Co-authored with Joachim D. Mueller
DOI: 10.1007/s00249-012-0792-x
Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two... more Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two proteins carrying distinct fluorescent labels. Because transiently transfected cells differ significantly in their expression level, the concentration ratio of the two expressed proteins varies, which in turn influences the measured fluorescence signal. Knowledge of the statistics of protein expression ratios is of considerable interest both from a fundamental point of view and for cellular fluorescence studies. Despite the perceived randomness of transient transfection, we were able to develop a quantitative model that describes the average and distribution of the protein expression ratio from a cell population. We show that the expression ratio is proportional to the molar plasmid ratio and relate the distribution to the finite number of active plasmids in the cell. The process of cationic lipid-mediated transfection is explored in more detail. Specifically, the influence of lipoplexes on the statistics of the expression ratio is examined. We further demonstrate that the transfection model provides a quantitative description of fluorescence fluctuation experiments, where only a fraction of the proteins are labeled.
Preparation and photo/chemical-activation of wormlike network micelles of core–shell quantum dots and block copolymer hybrids
by Meng Zhang
Zhang, M.; Rene-Boisneuf, L.; Hu, Y.; Moozeh, K.; Hassan, Y.; Scholes, G.; Winnik, M. A., J. Mater. Chem. 2011, 21 (26), 9692-9701.
Poly(styrene-b-4-vinylpyridine) diblock copolymers PS404-b-P4VP76 and PS317-b-P4VP76 (the subscripts indicate the... more
Poly(styrene-b-4-vinylpyridine) diblock copolymers PS404-b-P4VP76 and PS317-b-P4VP76 (the subscripts indicate the degree of polymerization) self-assemble into spherical ‘‘crew-cut’’ micelles with a PS core and P4VP corona when prepared in a mixture of chloroform and 2-propanol. When the micelles are formed in the presence of quantum dots (QDs), the nature of the structures formed depends upon the polymer and the type of QDs. In our previous report [Macromolecules, 2010, 43, 5066–5074], PS404-b-P4VP76 + CdSe QDs formed stable spherical hybrid micelles, but prolonged vigorous stirring of the solutions led to a rearrangement into wormlike networks and loss of photoluminescence (PL) from the QDs. Here we report that PS317-b-P4VP76 + CdSe/ZnS core–shell QDs behave differently.
Partial loss of PL intensity occurred upon addition of 2-propanol to the chloroform solution of the components, and the rearrangement to a network structure occurred spontaneously. We describe two strategies for recovery of the PL intensity for the QDs within the network, photo-activation and chemical activation with elemental sulfur.
Synthesis and PH-Responsive Properties of Pseudo-Peptides Containing Hydrophobic Amino Acid Grafts
by Rongjun Chen
Published in Journal of Materials Chemistry 2009
Pseudo-peptidic polymers have been synthesised by grafting L-valine (PV), L-leucine (PL) and L-phenylalanine (PP) onto... more Pseudo-peptidic polymers have been synthesised by grafting L-valine (PV), L-leucine (PL) and L-phenylalanine (PP) onto the pendant carboxylic acid moieties of a pH-responsive polyamide, poly(L-lysine isophthalamide). The pH-responsive aqueous solution properties of PV-75, PL-75 and PP-75 with a stoichiometric degree of substitution of 75 mol% have been compared with those of the parent poly(L-lysine isophthalamide) using UV-visible and fluorescence spectroscopy. At low concentrations (≤0.1 mg mL−1), the grafted polymers displayed pH-dependent conformation. The pH at the onset of hydrophobic association (pHh) and the pH range over which association occurred varied significantly between the different amino acid grafts. The pHh values of PV-75, PL-75 and PP-75 at 0.025 mg mL−1 were 6.2, 7.0 and 7.2, respectively. Increasing concentration enhanced intermolecular aggregation. A bis-functional Cy5 derivative, incorporated within the backbones of poly(L-lysine isophthalamide) (polyCy5) and PP-75 (PDP-75), was demonstrated to act as a fluorescence reporter on the state of polymer conformation and aggregation. The intracellular trafficking of PDP-75, examined by confocal microscopy, indicates potential applications of the grafted polymers in drug delivery and medical imaging.
New Monte Carlo model of cylindrical diffusing fibers illustrates axially heterogeneous fluorescence detection: simulation and experimental validation
by Tim Baran
Co-author: Thomas H. Foster
We present a new Monte Carlo model of cylindrical diffusing fibers that is implemented with a graphics processing... more We present a new Monte Carlo model of cylindrical diffusing fibers that is implemented with a graphics processing unit. Unlike previously published models that approximate the diffuser as a linear array of point sources, this model is based on the construction of these fibers. This allows for accurate determination of fluence distributions and modeling of fluorescence generation and collection. We demonstrate that our model generates fluence profiles similar to a linear array of point sources, but reveals axially heterogeneous fluorescence detection. With axially homogeneous excitation fluence, approximately 90% of detected fluorescence is collected by the proximal third of the diffuser for μs'/μa = 8 in the tissue and 70 to 88% is collected in this region for μs'/μa = 80. Increased fluorescence detection by the distal end of the diffuser relative to the center section is also demonstrated. Validation of these results was performed by creating phantoms consisting of layered fluorescent regions. Diffusers were inserted into these layered phantoms and fluorescence spectra were collected. Fits to these spectra show quantitative agreement between simulated fluorescence collection sensitivities and experimental results. These results will be applicable to the use of diffusers as detectors for dosimetry in interstitial photodynamic therapy.
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Quantitative analysis of multi-protein interactions using FRET: Application to the SUMO pathway.
by Sarah Martin
Protein Science 17:777-784
Sarah F. Martin, Michael H. Tatham, Ronald T. Hay, and Ifor D.W. Samuel
Protein–protein binding and signaling pathways are important fields of biomedical science. Here we report simple... more Protein–protein binding and signaling pathways are important fields of biomedical science. Here we report simple optical methods for the determination of the equilibrium binding constant Kd of protein–protein interactions as well as quantitative studies of biochemical cascades. The techniques are based on steady-state and time-resolved fluorescence resonance energy transfer (FRET) between ECFP and Venus-YFP fused to proteins of the SUMO family. Using FRET has several advantages over conventional free-solution techniques such as isothermal titration calorimetry (ITC): Concentrations are determined accurately by absorbance, highly sensitive binding signals enable the analysis of small quantities, and assays are compatible with multi-well plate format. Most importantly, our FRET-based techniques enable us to measure the effect of other molecules on the binding of two proteins of interest, which is not straightforward with other approaches. These assays provide powerful tools for the study of competitive biochemical cascades and the extent to which drug candidates modify protein interactions.
Diol Appended Quenchers for Fluorescein Boronic Acid
Chemistry - An Asian Journal, Volume 5, Issue 3, Pages 581 - 588
Fluorescein isothiocyanate is treated with 3-aminophenylboronic acid to provide a fluorescently tagged boronic acid... more Fluorescein isothiocyanate is treated with 3-aminophenylboronic acid to provide a fluorescently tagged boronic acid derivative which is used to assess Förster resonance energy transfer (FRET) quenching upon boronate ester formation with a series of bespoke diol appended quenchers. Fluorescence spectroscopy comparison of quenching efficiency between treatment of fluorescein and its boronic acid appended congener with quencher appended diol reveals boronate ester formation (covalently linked) to be the more efficient regime and from the panel of quenchers which also included nucleosides.
Boronic acid based photoinduced electron transfer (PET) fluorescence sensors for saccharides
A New Journal of Chemistry (advanced article)
A simple three step synthesis was developed to provide six novel modular sensors, consisting of three para sensors,... more A simple three step synthesis was developed to provide six novel modular sensors, consisting of three para sensors, and three meta sensors with naphthalene, anthracene and pyrene fluorophores. The interaction of the six sensors with the saccharides: D-glucose, D-fructose, D-galactose, and D-mannose, were evaluated. All sensors displayed increasing fluorescence intensity upon the addition of these saccharides, with all of the sensors showing enhanced selectivity for D-glucose over D-galactose, D-fructose and D-mannose. High affinity (Kobs) was also observed for the meta sensors with respect to the para sensors. The naphthalene and anthracene meta sensors showed particularly high affinity (Kobs) for D-galactose. Circular dichroism spectroscopy was used to probe the structures of the complexes formed. Cyclic complexes were formed between all six sensors and D-glucose. Whilst naphthalene and anthracene meta sensors which displayed high affinity for D-galactose also formed cyclic complexes with that saccharide.
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