Virus purification using 20% sucrose cushion centrifugation

Go to the profile of thomson90
Mar 22, 2018
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Hi, for those with experience, can you please help me check if anything wrong with my protocol?

- 900ul virus supernatant (clarified from cell debris by low speed centrifugation and 0.45um filter)
- layer on top of 100ul 20% sucrose in a 1.5ml tube
- spin max speed (13200rpm) with normal tabletop centrifuge machine for 4h (we do not have ultracentrifuge machine in our lab)
- pipet out supernatant, remove sucrose
- air dry the tube upside down in cabinet for 20 min
- resuspend in 100ul PBS
- recover in RT for 10 min

Take 10ul pelleted virion + 2.5 ul 4X Laemli buffer and run SDS-PAGE for western blot detection

thanks

1 Comments

Go to the profile of mdfenko
mdfenko 5 months ago

sorry for the late response, it looks like it should work, but don't throw away anything, just in case (in fact, you should use all fractions and interfaces on the western).
hopefully, there are enough g forces to push the virus through the sucrose.