Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from the primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester).
Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group of the linker lipid incorporated in the liposomes with EDC and sulfo-NHS, and then covalently conjugating the antibodies to the lipids through displacement of sulfo-NHS groups by antibody amines, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.
ImmunoFluor™-Succinyl is a non-PEGylated product. For other amine reactive (PEGylated and non-PEGyalated products) and also ImmunoFluor™ products suitable for other types conjugation methods see here.
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1、如果是提取的总蛋白，然后做WB，用β-actin或者GAPDH做内参肯定是没有问题的，这是公认的东西。
2、如果用膜蛋白提取试剂盒提取蛋白，再用β-actin作为内参似乎不妥，因为理论上来讲β-actin在膜上是不表达的。WB能做出β-actin来是因为膜蛋白提取时把胞质蛋白也提出来了。然而，如果我们试验目的是用药物处理细胞，比较处理前后某种膜蛋白的表达情况，此时用膜蛋白提取试剂盒提取膜蛋白后再用β-actin做内参似乎就不妥了，因为你根本不知道药物处理前后混杂了多少的胞质蛋白进来。如果没有胞质蛋白混进去的话，β-actin就是检测不到的。
当然做都是可以做，需要带上阴性和阳性的对照组，以区别出是抗体制备的问题还是抗原决定簇被破坏的问题。
第一，血清成分复杂，最好用密理博的Montage Albumin Deplete Kit去除血清里50%的白蛋白
第二，磷酸化蛋白提取蛋白的时候，最好加入原钒酸钠，和磷酸酶抑制剂，防止蛋白脱磷酸化。
第三，不要用牛奶做封闭剂，用BSA。因为牛奶中含有磷酸化酪蛋白，防止非特异带。
第四，如果条带杂带多，而且，条带弱，可以选择millipore的signal boost，增加条带特异性，而且，增强条带的亮度。
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