2 replies to this topic

[pathlily]

Hello!

I'm planning a massive cell culture time-course, from which I'd like to prepare cell lysates, in order to do westerns later.
I'm planning on scraping in RIPA buffer (with appropriate inhibitors added), then agitate in the cold or pipette up and down a bunch of times.

Most protocols then say that I should then spin to remove debris and then go on to perform a protein assay on the supernatant, followed by SDS-PAGE but don't indicate when would be a good time to stop and freeze/store.

What is the best time to freeze my samples (I'm going to be exhausted at sample collection point, so I'd prefer minimising the work at this stage).
Should I freeze before spinning or after?
I'll probably be freezing in a isopropanol/dry-ice mix; do you think that would be okay?


Thanks!

[bob1]

Either should be fine - the freeze/thaw should, in theory, help lyse the cells if you freeze before spinning.

dry ice freezing is fine.

[pathlily]

Counting down the hours until time zero: thanks for the advice!