protein bands are not migrating over to the whole gels. - please suggest me something, very urgent ! (Nov/26/2006 )
hi all,
its my great pleasure that i can put my problem with a hope that i will definitely get problem
troubleshooted.
i am running SDS-PAGE of mammalian protein but my protein bands are not moving to whole
length of gel. As the total length of my gel is 5.5 inch but my band are within 2.5 inch. i m confused
as i run the tracking dye up to full length.
can it be possible that dye move much faster.
please.... suggest something.
as i am in gr8 trouble.
ur help will be highly appreciated.
IMVHO you use too high gel concentration (ie. %T).
If you have any idea of MW range of your proteins you can pick right concentration of gel, check for details in some book about electrophoresis.
maybe your protein is insoluble and is stuck in the stacking gel..... 
does it move through the stacking?
Actually protein is coming out from the stacking gel. But problem is that bands are not resolving to that whole length of the gel. the are aggregating only at the top of gel.
i stop the gel after the dye come out of the gel.
if the stacking gel buffer is 8.8, then does it affect the mobility of protein.
is it possible that dye is moving with very high speed?
is there anybody who is resolving the hepg2 total protein on the gel ?
which gel percentage should be used.
thanx in advance.
colleagues in downer floor encounter relative same problem.
They fixed it by running at up to 120V in stacking (and then they go up to 250V in running part of gel but that's not necessary 
)
The pb came from the buffer used for pouring gel and the major was from running buffer.
these are first-to-chick things and i'm sorry as i can't speaak about more.
what is your electrode buffer? if it is tris-glycine then your stacking gel buffer should be pH 6.8. if it is tris-tricine or just tricine then you are using the wrong buffer for larger proteins.
which kind of buffer?tris glycine? it must be 6.8
if the stacking gel buffer is 8.8, then does it affect the mobility of protein.
which kind of buffer?tris glycine? it must be 6.8
when i cheked the pH of the stacking gel buffer it was more than 8. was my prob due to this?
yes i think so if its tris glycine buffer