[In vitro Kinase assay] Do phosphorylated proteins migrate more in SDS-PAGE? - (Nov/10/2007 )
Hi All,
Recently I performed in vitro kinase assay using gamma-P32[ATP] and sds-page subsequently. In 12% gel, my kinase (66kd) was autophosphorylated and appeared at ~30kd. My histone H1 (21.5kd) substrate was also phosphorylated and appeared at ~13kd. (There are only two bands in the film - at ~30kd and ~13kd)
When I used another substrate (112kd) with the same kinase (66kd), the phosphorylated 112kd substrate was appeared at ~65kd in 10% gel. 65kd band was the band of the strongest intensity and there were some background bands. But autophosphorylation did not seem to be obvious at this time.
When proteins are phosphorylated, do they migrate more like my in vitro kinase assay results? (66->~30kd, 21.5->~13kd and 112->~65kd)
Thank you so much in advance.
Hi,
I have a question. First, how are you doing the in vitro kinae assay? Are you adding recombinant proteins to cell extract with p32? If so, it is unlikely that the bands you see are the recombinant proteins you are including in the assay but some
proteins that are phosphorylated in the extract.
Usually when proteins are phosphorylated their migration shift slightly upwards by a few kDA (~2-5). A change of 30-60 kDA would be unheard of.
Recently I performed in vitro kinase assay using gamma-P32[ATP] and sds-page subsequently. In 12% gel, my kinase (66kd) was autophosphorylated and appeared at ~30kd. My histone H1 (21.5kd) substrate was also phosphorylated and appeared at ~13kd. (There are only two bands in the film - at ~30kd and ~13kd)
When I used another substrate (112kd) with the same kinase (66kd), the phosphorylated 112kd substrate was appeared at ~65kd in 10% gel. 65kd band was the band of the strongest intensity and there were some background bands. But autophosphorylation did not seem to be obvious at this time.
When proteins are phosphorylated, do they migrate more like my in vitro kinase assay results? (66->~30kd, 21.5->~13kd and 112->~65kd)
Thank you so much in advance.
Yes, I used all recombinant proteins. I incubated GST fused kinase with purified substrate (we purchased them from sigma/upstate). Then, what are the phosphorylated proteins?
I am not sure what the bands are bunch I have a suspicion. Do you do a control where you
perform the kinase reaction with just GST and not your GST-Kinase as
a negative control? I'm willing to bet that this would clarify alot of your issues.