During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge.
Copper-free click chemistry is a fairly new chemistry that has been commercialized during the past few years. More and more click chemistry-based reagents are becoming available commercially which makes the formulation development much easier for scientists. The great advantage of this chemistry is biocompatibility since no cytotoxic copper catalyst is required. By far, click chemistry is the most efficient and easiest conjugation chemistry available for coupling of antibodies and other reactive ligands to the surface of the liposomes. The conjugation chemistry is based on the reaction of the dibenzocyclooctyne (DBCO) reagent with an azide linker to form a stable triazole. DBCO moiety can be on the antibody and azide moiety can be on liposomes and vice versa. This conjugation protocol is based on the reaction of the dibenzocyclooctyne (DBCO) group of the liposomes with an azide linker on the antibody, peptide or proteins.
There are many commercialized reagents that can be used for azide modification of proteins, peptides and antibodies. To see the list of commercialized reagents for azide modification see here.
For other reactive (PEGylated and non-PEGyalated products) ImmunoFluor™ products suitable for other types conjugation methods see here.
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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。
转膜后就要区分与胶接触的一面与另一面了。话说楼主好奇的话可以跑蛋白的试试看哈。比如目的蛋白的胶用光滑面贴着胶，内参蛋白用粗糙面贴着胶一起转膜试试转膜结果看看呢。
1. 使用预染 Marker，不过分子量不是特别准；
2. 所使用的 Marker 条带与待测蛋白质带有相同的抗原表位，比如都带有 His 融合标签；
3. 可以使用普通的 Marker 转膜使用 丽春红 等染色，然后在膜上标记出 Marker 各条带的位置。
考马斯亮兰染液也可以重复用。新配的染液10分钟即可，重复3次后要染30分钟。
一抗二抗可以重复利用,但是注意要在5%milk中加入0.2% sodium azide ,并且用完以后放入4度保存,我的经验重复使用4-5次是肯定没有问题的.如果保存不当,就会污染微生物,只能丢弃.
