Western Blot/SDS-PAGE samples - boiled or not boiled? - (Nov/07/2007 )
I have another question.
What makes a sample necessary to be boiled?
And if I'm interested in testing an old sample (say which was already boiled in a previous experiment) should I boil it again prior to another SDS-PAGE? Or is it better to always start with a 'fresh' sample (say a sample which is just as old but has not been boiled)?
ps. I am looking at phosphorylation of AMPK in HepG2 cells.
-seriously a beginner at this kind of stuff-
If you have previously boiled the sample it should not be necessary to reboil it.
Boiling in SDS denatures the proteins and breaks the protein complexes so that you can more accurately seperate the proteins by weigh. You can run non- boiled ones, but you really need to be on native gels for that (when you're looking for activity or complexes) if you want meaningful results.
If you're looking at phosphorylation- make sure you're using cold buffers when extracting (on ice) and phosphatase inhibitors in your buffers (so that you prevent loss of phosphorylation after extraction). Good Luck, its not that difficult. Just remember to not leave the samples at 100C overnight (Like I did earlier this week) they really don't like that
One of those weeks
i was a bit confused since my phosphorylated samples needs to be boiled but the non-phosphorylated one does not.
yes everything was kept cold with ice and phosphatase inhibitor cocktails were used. thanks.