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Re: SDS-PAGE mobility phosphopeptides
The reference we use for gel shift is the one originally developed for ERK2
(Posada and Cooper, 1992, Science 255: 212-215). As with regular
acrylamide gels, we chose an appropriate acrylamide concentration to put
the band of interest in the middle 1/3 of the gel (so 7.5% for larger
proteins, 15% for smaller proteins). One of our post-docs thinks that its
best to aim for a slightly higher position, then run the lower molecular
weight components off the bottom, but I'm not convinced about that. The
stacking interface can be critical to seeing a good gel shift, so you might
need to try different methods of making your stacker and use the gel
immediately after polymerizing the stacker.
To see gel shifting in a protein of 300 kDa, is likely to be difficult, but
I'd first a shallow gradient gel, say 4-12%, on a fairly long gel, or try a
7% gel and run gel longer than usual (if you use fluorescently labeled
standards, you can keep track of what is going on better, but most people
determine the time empirically). I've seen shifts in proteins of this
size, on regular gels, so it should be possible to optimize the shift to
make it easier to visualize.
To the person who asked about why phosphorylation might make a protein run
faster on gels, I've only seen one instance of a phosphorylated protein
that ran faster than the unphosphorylated protein (lipocortin), and I've
never been convinced that the person wasn't seeing a proteolysis instead of
a gel shift. I'd make sure I could reverse the gel shift by phosphatase
before I said that the phosphorylation caused the faster mobility. I would
also radiolabel the sample and show that the lower band was the one that
had the phosphate (I've seen phosphorylated proteins split into two bands,
but both bands were radiolabeled--this can be due to gel artifacts or to
formation of disulfide bonds).
>>That is why a different amount of cross-linker is used in gels where one
>>is trying to detect gel shifts.
>I'd like to know what amount of cross-linker to use for detecting
>phosphoprotein shifts. Also, do you know whether this amount should vary
>for very large proteins (e.g. 300-400 kDa)? I would appreciate any advice
>you can give me on this matter!
>>>Is there anybody why phosphorylated protein migrates slowly than
>>>dephosphorylated protein on SDS/PAGE?
>> The major reason is that the phosphorylation induces a structure on
>>the protein. Migration through the pores of the gel depends on the protein
>>forming an "ideal" random coil--anything that perturbs this random coil
>>structure, such as phosphorylation or formation of a disulfide bond, will
>>cause the protein to run slower (or shift up). The specific pore structure
>>of the gel will affect the degree of gel shifting. That is why a different
>>amount of cross-linker is used in gels where one is trying to detect gel
>> The presence of phosphate can also prevent SDS from associating
>>with the protein properly, but this probably requires a lot of phosphate
>>added to be significant.