
Liposomes are extensively used to study the interaction of proteins, peptides and other molecules with the surface of a lipid membrane. One of the parameters that affects this interaction is the charge of the liposomal membrane. Liposomes are always made in aqueous environment and they are sized to the desired size in liquid state using various methods such as high-pressure extrusion through nano sized pore track etch membranes. Liposome without water is meaningless. In rare occasions, liposomes are freeze dried and proliposomes are formed in the presence of a lyoprotectant such as trehalose. Using a lyoprotectant is necessary in order to maintain the size of the liposomes after rehydration.
The fundamental structure of cell membranes is bilayers composed of phospholipids, and the vital function of the phospholipids in the membrane is to help keep it fluid and semi-permeable. Conventional glycerophospholipids have acyl chains attached to the sn-1 and sn-2 positions of the glycerol backbone via an ester bond. Ether lipids are a unique class of glycerophospholipids that have an alkyl chain attached to the sn-1 position by an ether bond (glycerol-ether lipids). In ether lipids, the alcohol group attached to the phosphate is generally choline or ethanolamine. Ether-linked phospholipids such as 1-alkyl-2-acyl-phosphatidylcholine and dialkylphosphatidylcholine are also found in the plasma and organelle membranes of mammalian species. Ether lipids form approximately 20% of the total phospholipid in mammals with different tissue distribution; brain, heart, spleen and white blood cells have the highest levels, while liver have a very little amount of ether lipids.
Studies on the formation and thermodynamic properties of ether-linked phospholipid bilayer membranes have indicated that in contrast to ester-linked phospholipid, the formation of the non-bilayer structure takes place spontaneously. This is attributed to the weaker interaction between polar headgroups in the ether-linked than that in the ester-linked phospholipids. It has also shown that the phase behavior of the ether-linked phospholipid bilayer membranes in ambient pressure is almost equivalent to that of the ester-linked phospholipid bilayer membranes under high temperatures and pressures, and the difference in the phase behavior decrease as the alkyl-chain length increases.
Due to distinctive properties of ether lipids, liposomes made from ether lipids exhibit very unique characteristics and performance: a) the ether bonds are more stable than ester linkages over a wide range of acidic or alkaline pH; b) stability properties of the liposomes is enhanced by bipolar lipids, and the saturated alkyl chains gives stability towards degradation in oxidative conditions; c) the unusual stereochemistry of the glycerol backbone enhance the resistance against the attacks by other organism phospholipases.
Lyophosome™ product catalog is composed of a large selection of freeze-dried liposomes with various types of lipids and wide range of zeta potentials and different properties. Lyophosome™ products should be used by scientists who understand liposome formulation and have the proper equipment to check the size, separate non-encapsulated drugs and do the proper assays. Freeze-dried liposomes cannot be used blindly.
Phospholipase A2 (PLA2) cannot hydrolyze the ether lipid liposomes. Diether lipids do not go through hydrolysis due to having an ether bond instead of an acyl bond and therefore to do that, they are a suitable candidate for experiments that needs to be performed at a higher temperature for an extended period of time. For more information about hydrolysis and oxidation of phospholipids see here.


Saturated diether lipids can neither be hydrolyzed nor oxidized.

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TUNEL的缺点是:1),操作要求高,组织样本需要固定(即使是培养细胞,也需要固定),不恰当的固定方法对实验结果影响很大,导致背景过高或者信号过弱,因此实验结果重复性不好;有的人花上半年做一个体内的TUNEL是一点都不奇怪的。2),大部分诱导凋亡的药物也引起DNA损伤,从而产生DNA断裂,易引入假阳性;3)凋亡晚期细胞基因组大量降解,导致TUNEL标记反而减少,因此此法反应的是早期凋亡比例,不能严格反应凋亡比例,属于半定量研究。
尽管有如此多缺点,但由于其是目前为数不多的能原位标记凋亡细胞的方法,因此用于组织体内凋亡研究仍然是首选。但体外细胞实验研究,很少用此法。
凋亡检测中,TUNEL并不是过时的方法,现在研究凋亡的文献仍然常见。而且相反,还比以前多一点,因为现在体内实验越来越多了,甚至线虫的凋亡研究,都用TUNEL。
凋亡研究中,的确有几种方法过时了,当然是因为有替代方案了,比如DNAladder,电镜。至于AnnexinV是不能用来和TUNEL一起评论的,前者用于细胞,而且主要是悬浮细胞,后者主要用于组织。虽然有人也做镜下的AnnexinV观察,TUNEL的细胞staining,但这都不是主流。
回到lz的原帖,的确如大家所言,做贴壁细胞,不推荐用TUNEL。如果是经典凋亡途径,只是确定比例,用最经典,最常用的subG1法即可,如果是不确定是否是凋亡,用AnnexinV,不过贴壁细胞用此法,要注意消化时间。
那为什么SFDA不批准CA199CEAAFP等检测试剂盒作为癌症检测的手段呢?
而放射性核素标记,是对体外诊断试剂的某些元素进行放射性特征性标记,便于检测而已,这类的放射性强度不大,危害不高
在弱碱性(pH 8~9)、暗处、室温或40℃条件下,氨基酸的α-氨基很容易与2,4-二硝基氟苯(缩写为FDNB或DNFB)反应,生成黄色的2,4-二硝基苯氨基酸(dinitrophenyl amino acid,简称DNP-氨基酸)。多肽或蛋白质的N-末端氨基酸的α-氨基也能与FDNB反应,生成一种二硝基苯肽(DNP-肽)。由于硝基苯与氨基结合牢固,不易被水解,因此当DNP-多肽被酸水解时,所有肽键均被水解,只有N-末端氨基酸仍连在DNP上,所以产物为黄色的DNP-氨基酸和其它氨基酸的混合液。混合液中只有DNP-氨基酸溶于乙酸乙酯,所以可以用乙酸乙酯抽提并将抽提液进行色谱分析,再以标准的DNP-氨基酸作为对照鉴定出此氨基酸的种类。因此2,4-二硝基氟苯法可用于鉴定多肽或蛋白质的N-末端氨基酸。