Fluorescent proteins have become extremely popular tools for in vivo imaging as well as the study of localization,
motility and interaction of proteins in living cells. Bimolecular fluorescence complementation (BiFC) analysis based on
fluorescent proteins enables direct and high throughput visualization of protein-protein interactions in living cells. Two
red Bimolecular Fluorescent Complementation (BiFC) systems based on mRFP variants have been reported. However,
some physical-chemical characteristics of mRFP limited their applications, such as low pH-stability, relative low
brightness and low maturation rate. We have developed a new red BiFC system based on TagRFP, a novel monomeric
red fluorescent protein with high brightness, complete chromophore maturation, prolonged fluorescence lifetime and
high pH-stability. In this study, bFos and bJun were used as the positive protein-protein interaction pair, a mutant of bFos
(bFos) and bJun were used as the negative protein-protein interaction pair. bFos/ΔbFos was fused to N-terminal fragment
of TagRFP, and bJun was fused to C-terminal fragment of TagRFP. The BiFC systems based on TagRFP was confirmed
in living mammalian cells. Furthermore, the BiFC based on TagRFP allow analyzing multi-protein interactions when
combined with other BiFC systems. Thus, the BiFC based on TagRFP is very useful for investigating the complicated
and significant molecular mechanisms of multi-protein complex in living cells.
Benzisothiazdone, a kind of safe and effective ketone containing heterocycle, was introduced as an enhancer to
investigate the effect on optical clearing of the piglet skin with and without stratum corneum in vitro. The skin samples
were treated with different agents, such as, pure polyethyleneglycol 400, mixed solution (benzisothiazdone combining
with polyethyleneglycol 400) and isotonic saline. The total transmittance of samples was monitored by a single
integrating sphere system. The results show that the saline does not affect the rate of optical clearing obviously. For the
sample with stratum corneum, single optical clearing agent such as PEG400 penetrates slowly because of the barrier
function of SC. The stratum corneum makes the effect of optical clearing on the sample inconspicuous. In contrast, the
mixed solution can damage the barrier function of SC to some extent. For the sample without stratum corneum, an
obvious increase in the rate of optical clearing is in the single PEG400 administration stage, this effect will be more
remarkable if the Benzisothiazdone was used as an enhancer. We can conclude that SC can prevent skin from effecting
of single OCA, but the barrier will become weaker if right immersion agent is adopted as the enhancer.
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