eluting protein from the gel band... - (May/21/2006 )
from Pierce procedure i will elute the protein by incubaing it overnight at 30C in Tris/NaCL/EDTA buffer....but my protein is highly degradable....do you think it will not degrade during this? 
,...i am thinking of adding PMSF ...what would you suggest? thank you for your input...
unless you have some source of protease present (conaminated water? overlapping bands?) then pmsf should have no effect on the stability of your protein (unless it is a protease that can autolyse and is inhibited by pmsf). a little (1-2mM) dtt might help by maintaining cys and met in the reduced state.
if you are concerned about the length of time you will be incubating your protein then you can homogenize your gel slices in the buffer. afterwards, you pellet the gel and collect the supernate.
if you are concerned about the length of time you will be incubating your protein then you can homogenize your gel slices in the buffer. afterwards, you pellet the gel and collect the supernate.
mdfenko, thank you very much for your reply....so i will incubate in MilliQ autoclaved water...and since im extracting the band from urea-PAGE i will add 7M urea so that my protein will not aggregate....(it aggregates without urea...)
sounds like a plan.
keep in mind that the urea will decompose over time when incubating at or above room temperature. so your protein may come out of solution during the elution.
also remember that you will be eluting the gel buffer (tris-glycine?), so your solution will still be buffered (to a degree) and should be taken into consideration when planning the next step.
keep in mind that the urea will decompose over time when incubating at or above room temperature. so your protein may come out of solution during the elution.
also remember that you will be eluting the gel buffer (tris-glycine?), so your solution will still be buffered (to a degree) and should be taken into consideration when planning the next step.
thank you for your advice!
...so ill try at 30 overnight, but if my protein precipitates ill try room temperature instead....but about the buffers Tris-glycine is my running buffer....will it be in the band? i thought that rather the buffers i used to make up the gel.... .... ....the buffers in the gels are displaced by the reservoir buffer during electrophoresis (that's how stacking works). on some gel systems you can see the buffer front as it passes through the gel (slowly). that's also why the gel running pH is different from that of the running gel buffer.
thank you very much for your explanations....im so sorry im such an non-educated newby... 
your welcome. i appreciate the opportunity to show off.
don't be too hard on yourself. you learn these things as you go along. i'm happy that now there are forums where you can learn these things sooner rather than later and where you can share your knowledge and experience.