Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.
Gas-phase absorption spectroscopy using a
3.39μm
He–Ne laser enables the concentration of methanol vapor in a vapor-fed direct methanol fuel cell (DMFC) to be measured in real-time and in situ once high noise levels of the laser are corrected. The changes that methanol vapor concentrations bring to cell performance can be measured under various galvanostatic and potentiostatic conditions and analyzed with regard to methanol crossover and methanol transport capability. The absorption coefficient of +methanol vapor,
0.005336cm−1Torr−1
, is nearly constant under the DMFC operating conditions at a wavelength of
3.39μm
.
A theoretical model of solidification of a thermoset coating was constructed to examine the effects of a depthwise gradient in curing and skin formation on the coating's wrinkling behavior. The effects of coating thickness and catalyst concentration on the wrinkle wavelength of an acrylic-melamine coating were investigated with the model and mechanical profilometry. The model predicted that thin coatings with low and high catalyst concentrations gel quickly from top downward. Such coatings therefore have little time for the liquid below to diffuse into the already gelled skin to produce enough compressive stress to wrinkle the skin. The predicted time for complete gelation, as well as measured wrinkle wavelength in thicker coatings, rose as coating thickness was increased and showed an optimum as catalyst concentration was raised. Although time for complete gelation does not directly correlate with the wrinkle wavelength, the match of the trends of experimentally observed and theoretically predicted behavior is evidence that the essentials have been identified and represented by the model.
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