Dear All,
I use HEK293-T cells for preperation.
I treat them 24h with epoxomicin. And then I isolate proteins using RIPA buffer.
I precipitate overnight 5 mg protein with 25 micro liter of ubiquitin pan resin (N2510, nanotag).
I pellet the resin by centrifugation and wash it with 1 ml RIPA buffer, 4 times. So There is no real possibility to have residual non ubiquitinated protein.
I heat-up the resin using laemlii sample buffer and load it to western blot gel.
Then I blot using p53 antibody (sc-98).
My result:
I have 4 wells
1- hek lysate
2- hek treated with epoxymicin lysate
3- ubiquitin selector O/N precipitated HEK lysate
4- ubiquitin selector O/N precipitated epoxymicin treated HEK lysate
after p53 blot I see increased p53 in epoxymicin treated sample versus control ( 2 vs 1)
epoxy treated ubiquitin pulled-down p53 is higher than non-treated pulled down ( 4 vs3)
But the band that is coming at 3 and 4 and supposed to be ubiquitinated is at the same size with p53 from 2 and 1 ( 4 and 3 are at the same size with 1 and 2)
what can be the reason behind that ? Why I dont see a higher band for p53 after ubiquitin selection.
Thanks
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