E. coli and SDS-PAGE
bjd12 at cus.cam.ac.uk
Fri May 20 02:13:27 EST 1994
Timothy Bushnell (bushnt at rpi.edu) wrote:
: In an undergraduate lab, we are having the students prepare protein
: profiles of a stationary phase E. coli culture using SDS-PAGE. Currently,
: we spin 50 mls of culture down and resuspend the pellet in 1 ml of 62.5 mm
: Tris pH 6.8, 2% SDS, 5% B-mercapt. and 10% glycerol. This is then boiled
: for 5 to 10 minutes and the samples then applied to a SDS-PAGE gel.
: The problem is that the sample is very viscous and near impossible to
: successfully load on a gel. Typically, after the sample is loaded into the
: wells, and the pipet tip removed, the sample sticks to the tip and is
: removed as a gob into the upper tank buffer. Very rarely, when a sample is
: overboiled (20+ minutes) and loaded hot, we avoid this problem.
When I'm loading E.Coli from expression tests, I use 1mL of cell
culture, spun, resuspended in 100uL buffer (similar to yours), and boiled
for 20 min ... I only get away from the problem you describe when I boil for
this long, and the samples seem fine after it. Then the samples are spun
hard for 5 min, and 5-10uL loaded onto the gel. This works fine, and gives
good, clearly resolved gels.
I suspect its a combination of the long boil and the hard spin that
makes it work. Hope this helps,
: Tim Bushnell
: Rensselaer Polytechnic Institute
MRC Protein Function and Design,
"They can make me do it, but they can't make me do it with dignity."
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