Does phosphorylation affect protein migration in SDS-PAGE gel? - (Dec/24/2007 )

Dear Experts,

Recently I am running a Western blotting of a protein known to have many phosphorylation sites and sometimes I see two closely located bands around the expected position. I wonder whether one band (the bigger) represents the phosphorylated protein. I heard that phosphorylation slows protein migration, is that right?

Another thing is that in one cells, sometimes there is only one band, sometimese there are two bands, although the treatment remains the same. I wonder what may cause the variation in phosphorylation during cell treatments (miRNA transfection) or Western blotting procedures.

Thank you for your thoughts.

I think it should be the other way around. Phosphorylated proteins migrate faster than unmodified ones due to extra anionic charges these derivatives have. Many treatments can activate protein kinases. The phosphate groups may be cleaved off by phosphatase if you dont use inhibitors in your cell lysate.

hi,
in my experience phosphorylated proteins runs slower due to their higher molecular weights. whether u can see difference between phophorylated and non phosphorylated (total) proteins is purely depends on number of phosphorylation sites in protein and %of the gel and resolving time.

i wud not agree with genehunter-1 regarding his assumption about "phosphorylated protein run faster than total proteins". as the native protein charge (either anionc or cataionc) will be brought to highly negative charge with high conc of SDS in the sample buffer.
it is barely convincing that negative charge of phosphate group overcome the SDS charge and adds speed to the phosphorylated proteins.

welcome for comments

sravan payeli

You are right, phosphorylated proteins generally run slower than the native ones.

I really thank you guys for your thoughtful discussion. Sometimes I see stronger and smaller bands than the expected one. I guess the inconsistency (sometimes one band, sometimes two bands) may be caused by technical variations such as whether phosphatase inhibitors have been adequately added, etc. Are there any other causes for the inconsistency?

you could also be seeing the effect of various proteases on the sample (prior to addition of loading solution).

hi,
just to minimize most of the variations, as soon as u finish the experiment, harvest lysates, estimate protein and add the sample buffer to whole cell lysate and boil it.
then u can aliquote 10 or 20ug of total protein per vial and freeze according to ur western blot protocol.

i know it is a tedious process, but for sensitive proteins it may pay off for your efforts.

sravan

one way to determine if the top of the two bands is due to phosphorylation is to treat some of your lysate with alkaline phosphatase- you should lose the phosphorylated bands-

Normally your protein samples should run slower because of the heightended mw, but if you have only one or two more phosphorylations added to your unphosphorlyated protein, you can not distinguish this in a normal SDS-gel of 10 or 15%, you would need a very high resolution for this small dfference. Perhaps what you see are two isoforms of one proteins with somewhat different sizes. Western blotting with phospho-specific antibodies can help as well as dephosphorylation of your proteins.