I am using Phos-tag gels for a while now and generally get good results. This time, however, I tried running a Ser-to-Glu mutated protein, where the Ser is usually phosphorylated. I thought I would just lose the phospho-band, but instead it shifted. Has anyone done something similar and could tell me their results? I thought unphosphorylated amino acids do not react with the Phos-tag.
Hi,
Is it possible that some other site is phosphorylated in your protein and hence the shift?
Ser-to-Glu change is for mimicking phophorylated protein by adding stable charge using Glu... of course it is not the same as a phopho-group. if you want to investigate non-phospho form, you should use Ser-to Ala change. that is my first impression.
Hi Sanjana, yes, I think there is another site phosphorylated. But this would also be phosphorylated in the non-mutant and the shift is different. Attila, I have also done a Ser-to-Ala mutation and do not see a shift. It is complicated... So, probably two sites phosphorylated, site A changed to Ala - no difference to non-mutant, site A changed to Glu, shift (faster migration). Of course, all these are shifts compared to the unphosphorylated form. I would like to hear what others saw with a Ser-to-Glu on a Phos-gel?
if you suppose to have another phospho site, which is currently distributing on mutated proteins, use alkaline phosphatase treatment in that samples as it is suggested in the protocol. Good luck.
Well. I have used lambda phosphatase on the non-mutated one and the band vanishes. So, this is due to phosphorylation. You say to use phosphatase on the Ser-to-Glu? Good idea, I do that. Thanks for your input, you two!
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology