超离提取的exosome WB鉴定

luckkit

xuemei7868650 发表于 2015-2-13 17:48
同求exosome蛋白的提取方法

就是买试剂盒分离Exosomes,如果想进一步MS鉴定的话，就跑2D

luckkit

转来的，
https://www.researchgate.net/pos ... nvolving_exosomes/1
Ketil winther Pedersen · 31.11  · 59.54  · ThermoFischer Scientific,


Hi Sami, I would just like to share some thoughts with you after working with exosomes and downstream western blotting for a while. It is my experience that there are many factors that are important for WB detection. First, its the source from where the exosomes are derived from. It seems that different host cells release different amounts of exosomes into the surrounding environment. If cell lines are the source - for how long the cell culture has been "running" (# of splits) might have an impact on the amount of exosome released. Then it is the enrichment step. How is the exosomes enriched? Ulracentrifugation? Precipitation? I did spend a lot of time optimizing the amounts of exosomes used and also dilutions used and also lysis conditions (happy to share that with you. just send me an email to ketil.pedersen@lifetech.com). Then, its the blotting part of the workflow. How is the proteins blotted to the PVDF membranes? wet blotting? Finally, its the labeling. Have you tested whether you are able to detect the classical tetraspanin proteins such as CD63 or CD81. If the exosomes should express these proteins it could be a way to verify that exosomes are in your enriched sample (alternatively you might consider analyzing the enriched exosome prep by negative stain electron microscopy just to verify the presence of exosomes). Labeling for CD63/CD81 usually require non-reducing conditions as Dr. Ana mentioned above. In terms of exposing the bands my experience is that chemiluminescence is required to visualize the target bands.

good luck
Ketil Winther Pedersen