Gel shifts: run them fast or slow?
Marieke R. Koedood Zhao
rkoedood at bio.bu.edu
Mon Dec 16 13:12:49 EST 1996
Depends on your specific complexes. Try out the 'recommended' conditions,
and then change if it doesn't come out right.
I remember running a loooong gel to resolve certain complexes. Many hours,
recycling the buffer every two hours, only to find my band being too
diffuse to be usable for publication. So, I had to settle for shorter gels
with that non-specific band closer to my band than I was happy about.
just some ideas...
Marieke Koedood Zhao
John Pawlowski (johnp at qadas.com) wrote:
: Hi all,
: I am interested in getting the best possible resolution in gel shift-
: assays in which radiolabelled oligonucleotide probes are incubated with
: protein extracts from cells, and run under non-denaturing conditions. A
: particular question I have is whether or not the gels should be run fast
: (approx 2 hours for 15 cm gel) or slow (overnight). It seems to me that
: running the gels fast (applying a higher current) might "strip" away the
: probe from the bound protein due to charge differences between the probe
: and the protein. On the other hand, it seems to me that applying a lower
: current and running the gel slowly would give the oligonucleotide
: probe/protein complexes more time to disassociate in the gel, leading to
: poorer resolution. All thoughts on these questions are greatly
: appreciated, as well as any other advice on performing high-quality gel-
: shift experiments. Thanks much.
: John Pawlowski
: johnp at qadas.com
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