To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging. 相似文献
Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. (31)P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is approximately 50K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid (13)C'-(31)P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular (13)C-(31)P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced (31)P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes. 相似文献
Uniformly sized microparticles of poly(d,l-lactic-co-glycolic) (PLGA) acid, with controllable median diameters within the size range 40–140 μm, were successfully prepared by membrane emulsification of an oil phase injected into an aqueous phase, followed by solvent removal. Initially, simple particles were produced as an oil in water emulsion, where dichloromethane (DCM) and PLGA were the oil phase and water with stabiliser was the continuous phase. The oil was injected into the aqueous phase through an array type microporous membrane, which has very regular pores equally spaced apart, and two different pore sizes were used: 20 and 40 μm in diameter. Shear was provided at the membrane surface, causing the drops to detach, by a simple paddle stirrer rotating above the membrane. Further tests involved the production of a primary water in oil emulsion, using a mechanical homogeniser, which was then subsequently injected into a water phase through the microporous membrane to form a water in oil in water emulsion. These tests used a water-soluble model drug (blue dextran) and encapsulation efficiencies of up to 100% were obtained for concentrations of 15% PLGA dissolved in the DCM and injected through a 40 μm membrane.
Solidification of the PLGA particles was followed by removal of the DCM through the surrounding aqueous continuous phase. Different PLGA concentrations, particle size and osmotic pressures were considered in order to find their effect on encapsulation efficiency. Osmotic pressure was varied by changing the salt concentration in the external aqueous phase whilst maintaining a constant internal aqueous phase salt concentration. Osmotic pressure was found to be a significant factor on the resulting particle structure, for the tests conducted at lower PLGA concentrations (10% and 5% PLGA). The PLGA concentration and particle size distribution influence the time to complete the solidification stage and a slow solidification, formed by stirring gently overnight, provided the most monosized particles and highest encapsulation efficiency. 相似文献