preparing samples for phosphorylation sites identification - (Jan/09/2008 )
hi,
I desperately need someone to clarify me briefly a process of phosphorylation sites identification.
My plan is to transfect cells of interest with my gene, then collect cell lysates, do 1D SDS-PAGE, cut the band and sent it to service where they do MS etc.
I'm completely green in that field. I search literature but now I have a chaos in my head. Everyone uses another method.
Do my gene need any tag? Then which tag is the best (V5, His, FLAG, GST)? Do I have to prepare samples before PAGE in any special manner? I know I have to add all these inhibitors to protect against dephosphorylation.
Should I get rid of SDS before I sent band to Mass Spec service?
Thank you in advance
OK, I have no experience in sequencing, but I know you will never be able to sequence anything if you don't have a pure band.
In these conditions, it's impossible to get a band that contains only your protein. There are several proteins with the same or a narrow molecular weight.
it would be better to have a tagged protein, so you could purify it.
I think that his tag is fine. you can use a Ni column to purify your protein. Then, check the purity on a gel, but i guess it will still not be complete pure. you will see several thin bands.
I would then cut the band of interest and send it to MS (after purification, the band of interest should be now quite pure)
I desperately need someone to clarify me briefly a process of phosphorylation sites identification.
My plan is to transfect cells of interest with my gene, then collect cell lysates, do 1D SDS-PAGE, cut the band and sent it to service where they do MS etc.
I'm completely green in that field. I search literature but now I have a chaos in my head. Everyone uses another method.
Do my gene need any tag? Then which tag is the best (V5, His, FLAG, GST)? Do I have to prepare samples before PAGE in any special manner? I know I have to add all these inhibitors to protect against dephosphorylation.
Should I get rid of SDS before I sent band to Mass Spec service?
Thank you in advance
which band should be cut out? the product of the transfected gene? you need no tag but has to identify clearly the polypeptide (f.i. in a Western blot); better use 2D as a band in 1D contains often several polypeptides...
nrmally no tag is needed. A strong overexpression is recommended, but you may also do 2 lanes, one submitted to western blot and the other one for coomassie and ms analysis.
It's essential that you use NaF or phosphatase inhibitor cocktails during protein isolation, and you may add NaF (relative cheap) to all reagents.