Cellsome®-Natural PC extract catalog is composed of many liposomes made from phosphatidylcholine (PC) lipids that are made from natural lipids that are extracted from egg yolk, soybean, tissues and organs such as brain, heart and liver with and without cholesterol. Adding 30% cholesterol makes the liposomes serum stable and is necessary when the liposomes are used in the presence of serum.
Liposomes made from egg PC, contain both saturated and unsaturated lipids. These liposomes contain0.2% of the 14:0 lipid, 32.7% 16:0 lipid, 12.3% 18:0 lipid, 1.1% 16:1 lipid, 32% 18:1 lipid, 17.1% 18:2 lipid, 0.2% 20:2 lipid, and 0.3% 20:3 lipid, 2.7% 20:4 lipid and 0.4% 22:6 lipid.
Liposomes made from Brain PC contain both saturated and unsaturated lipids. They contain 0.3% 14:0 lipid, 30.6% 16:0 lipids, 16.5% 18:0 lipid, 0.9% 16:1 lipid, 33.3 % 18:1 lipid, 1.1% 18:2 lipid, 0.8% 20:1 lipid, 0.2% 20:3 lipid, 3.1% 20:4 lipid and 0.6% 22:6 lipid.
Liposomes made from Heart PC also contain 40% plasmalogen ether lipid. Ether lipids are more stable and do not get hydrolyzed. Heart PC also contains saturated and unsaturated lipids. It contains about 23% 16:0 lipid, 7% 18:0 lipid, 11% 18:1 lipid, 43% 18:2 lipid and 6% 20:4 lipid.
Liver PC also contains saturated and unsaturated lipids. Almost half of the lipids are saturated and the other half unsaturated. It is composed of 14% 16:0 lipid, 33% 18:0 lipid, 17% 18:1 lipid, 12% 18:2 lipid, 8% 20:3 lipid and 7% 20:4 lipid.
Soy PC also contains saturated and unsaturated lipids. Soy PC is mostly composed of unsaturated lipids and have a larger percentage of unsaturated PCs compare to egg, brain, heart and liver extracted PCs. Liposomes made from Soy PC contain 14.9% 16:0 lipid, 3.7% 18:0 lipid, 11.4% 18:1 lipid, 63% 18:2 lipid and 5.7% 18:3 lipid.
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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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