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各位大神 我用了life和SBI的试剂盒提血清的exosomes感觉总是有问题
SBI说明书后边有一个做western的protocol
1. If frozen, thaw culture media or urine sample on ice
2. Combine 500µl serum + 120 µl ExoQuick
3. Mix well by inversion three times
4. Place at 4ºC for 30 minutes (or up to12 hours)
5. Centrifuge at 1500 × g for 30 minutes
6. Remove supernatant, keep exosome pellet
7. Centrifuge at 1500 × g for 5 minutes to remove all traces of fluid (take great care not to disturb the pellet)
8. Add 200 µl RIPA buffer to exosome pellet and vortex 15 seconds
9. Place at room temperature for 5 minutes (to allow complete lysis)
10. Add Laemmli buffer (with Beta-mercaptoethanol) and heat at 95⁰C for 5 minutes.
11. Chilled on ice for 5 minutes before loading onto gel
12. Perform standard SDS-PAGE electrophoresis and Western transfer onto PVDF membrane
13. Block with 5% dry milk in Tris Buffered Saline + 0.05% Tween (TBS-T) for 1 hour
14. Incubate blot overnight at 4°C with SBI's exosome specific antibody (e.g. CD9) at 1:1000 dilution (5% dry milk in TBS-T)
15. Wash 3X with TBS-T
16. Incubate one hour at room temperature with SBI's Goat anti-Rabbit-HRP antibody at 1:20,000 dilution (5% dry milk in
TBS-T)
17. Wash 3X with TBS-T
18. Incubate blot with chemi-luminescence substrate and visualize on film or other imaging equipment
我基本上是按照说明书做的 但是做了很多次都没有结果 请问500ul血清能不能提出eoxosmes？加入试剂之后会看到明显的黄色沉淀 用RIPA裂解液可以重悬 但感觉好像不能完全裂解 有没有必要再离心一下去除沉淀？有没有什么注意事项求指点 试剂盒都快用完了 一个条带都木有 谢谢！ |
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|Archiver|手机版|外泌体之家 | exosomes & microvesicles
发表于 2015-3-13 15:26:33
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发表于 2015-3-18 10:04:30
